Neutralizing anti-sars-cov-2 antibodies

ABSTRACT

This disclosure provides novel neutralizing anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof. The disclosed anti-SARS-CoV-2 antibodies constitute a novel therapeutic strategy in protection from SARS-CoV-2 infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 63/199,676, filed Jan. 15, 2021. Theforegoing application is incorporated by reference herein in itsentirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under grant nos.P01-AI138398-S1 and 2U19AI111825 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to antibodies directed to epitopes ofSARS-CoV-2 Coronavirus 2 (“SARS-CoV-2”). The present invention furtherrelates to the preparation and use of neutralizing antibodies directedto the SARS-CoV-2 spike (S) glycoproteins for the prevention andtreatment of SARS-CoV-2 infection.

BACKGROUND

SARS-CoV-2 is the virus that causes coronavirus disease 2019 (COVID-19).It contains four structural proteins, including spike (S), envelope (E),membrane (M), and nucleocapsid (N) proteins. Among them, S protein playsthe most important role in viral attachment, fusion, and entry, and itserves as a target for development of antibodies, entry inhibitors, andvaccines. The S protein mediates viral entry into host cells by firstbinding to a host receptor through the receptor-binding domain (RBD) inthe 51 subunit and then fusing the viral and host membranes through theS2 subunit. SARS-CoV and MERS-CoV RBDs recognize different receptors.SARS-CoV recognizes angiotensin-converting enzyme 2 (ACE2) as itsreceptor, whereas MERS-CoV recognizes dipeptidyl peptidase 4 (DPP4) asits receptor. Similar to SARS-CoV, SARS-CoV-2 also recognizes ACE2 asits host receptor binding to viral S protein. SARS-CoV-2 has infected 45million individuals and is responsible for over 1 million deaths todate. There is a pressing need for agents for treating or preventingSARS-CoV-2 infection.

SUMMARY

This disclosure addresses the need mentioned above in a number ofaspects by providing neutralizing anti-SARS-CoV-2 antibodies orantigen-binding fragments thereof.

In one aspect, this disclosure provides an isolated anti-SARS-CoV-2antibody or antigen-binding fragment thereof that binds specifically toa SARS-CoV-2 antigen. In some embodiments, the SARS-CoV-2 antigencomprises a Spike (S) polypeptide, such as a S polypeptide of a human oran animal SARS-CoV-2. In some embodiments, the SARS-CoV-2 antigencomprises the receptor-binding domain (RBD) of the S polypeptide. Insome embodiments, the RBD comprises amino acids 319-541 of the Spolypeptide.

In some embodiments, the antibody or antigen-binding fragment thereof iscapable of neutralizing a plurality of SARS-CoV-2 strains.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises: three heavy chain complementarity determining regions (HCDRs)(HCDR1, HCDR2, and HCDR3) of a heavy chain variable region having theamino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123,125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151,153, 155, 157, 159, 161, 163, 165, or 167; and three light chain CDRs(LCDR1, LCDR2, and LCDR3) of a light chain variable region having theamino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 158, 160, 162, 164, 166, or 168.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises: (i) a heavy chain variable region having an amino acidsequence with at least 75% identity to one selected from those in Table2 or 4; and/or (ii) a light chain variable region having an amino acidsequence with at least 75% identity to one selected from those in Table2 or 4.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises: (i) a heavy chain variable region having the amino acidsequence of one selected from those in Table 2 or 4; and/or (ii) a lightchain variable region having the amino acid sequence of one selectedfrom those in Table 2 or 4.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises a heavy chain variable region and a light chain variableregion that comprise the amino acid sequence pair selected from those inTable 2 or 4.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises: a heavy chain variable region having an amino acid sequencewith at least 75% identity to the amino acid sequence of SEQ ID NO: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, or167; or having the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143,145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, or 167; and alight chain variable region having an amino acid sequence with at least75% identity to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, or 168; orhaving the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166, or 168.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises a heavy chain variable region and a light chain variableregion comprise the respective amino acid sequences of SEQ ID NOs: 1-2,3-4, 5-6, 7-8, 9-10, 11-12, 13-14, 15-16, 17-18, 19-20, 21-22, 23-24,25-26, 27-28, 29-30, 31-32, 33-34, 35-36, 37-38, 39-40, 41-42, 43-44,45-46, 47-48, 49-50, 51-52, 53-54, 55-56, 57-58, 59-60, 61-62, 63-64,65-66, 67-68, 69-70, 71-72, 73-74, 75-76, 77-78, 79-80, 81-82, 83-84,85-86, 87-88, 89-90, 91-92, 93-94, 95-96, 97-98, 99-100, 101-102,103-104, 105-106, 107-108, 109-110, 111-112, 113-114, 115-116, 117-118,119-120, 121-122, 123-124, 125-126, 127-128, 129-130, 131-132, 133-134,135-136, 137-138, 139-140, 141-142, 143-144, 145-146, 147-148, 149-150,151-152, 153-154, 155-156, 157-158, 159-160, 161-162, 163-164, 165-166,or 167-168.

In some embodiments, the antibody or antigen-binding fragment thereof isa multivalent antibody that comprises (a) a first target binding sitethat specifically binds to an epitope within the S polypeptide, and (b)a second target binding site that binds to a different epitope on the Spolypeptide or a different molecule. In some embodiments, themultivalent antibody is a bivalent or bispecific antibody.

In some embodiments, the antibody or the antigen-binding fragmentthereof further comprises a variant Fc constant region. In someembodiments, the antibody is a monoclonal antibody. In some embodiments,the antibody is a chimeric antibody, a humanized antibody, or ahumanized monoclonal antibody. In some embodiments, the antibody is asingle-chain antibody, Fab or Fab2 fragment.

In some embodiments, the antibody or antigen-binding fragment thereof isdetectably labeled or conjugated to a toxin, a therapeutic agent, apolymer, a receptor, an enzyme, or a receptor ligand. In someembodiments, the polymer is polyethylene glycol (PEG).

For example, an antibody of the invention may be coupled to a toxin.Such antibodies may be used to treat animals, including humans, that areinfected with the virus that is etiologically linked to SARS-CoV-2. Forexample, an antibody that binds to the spike protein of the coronavirusthat is etiologically linked to SARS-CoV-2 may be coupled to a tetanustoxin and administered to an animal suffering from infection by theaforementioned virus. The toxin-coupled antibody is thought to bind to aportion of a spike protein presented on an infected cell, and then killthe infected cell.

An antibody of the invention may be coupled to a detectable tag. Suchantibodies may be used within diagnostic assays to determine if ananimal, such as a human, is infected with SARS-CoV-2. Examples ofdetectable tags include fluorescent proteins (i.e., green fluorescentprotein, red fluorescent protein, yellow fluorescent protein),fluorescent markers (i.e., fluorescein isothiocyanate, rhodamine, texasred), radiolabels (i.e., 3H, 32P, 1251), enzymes (i.e.,(3-galactosidase, horseradish peroxidase, β-glucuronidase, alkalinephosphatase), or an affinity tag (i.e., avidin, biotin, streptavidin).

In another aspect, this disclosure provides a pharmaceutical compositioncomprising: the antibody or antigen-binding fragment thereof of any oneof the preceding claims and optionally a pharmaceutically acceptablecarrier or excipient.

In some embodiments, the pharmaceutical comprises two or more of theantibody or antigen-binding fragment thereof described above, such asany combinations of the antibody or antigen-binding fragment thereofcomprising a heavy chain and a light chain that comprise the respectiveamino acid sequences of one selected from those in Table 2 or 4.

In some embodiments, the two or more of the antibody or antigen-bindingfragment thereof comprise: (1) a first antibody set comprising: (i) afirst antibody or antigen-binding fragment thereof comprising a heavychain variable region and a light chain variable region comprising therespective amino acid sequences of a first antibody selected from thosein Table 2 or 4; and (ii) a second antibody or antigen-binding fragmentthereof comprising a heavy chain variable region and a light chainvariable region comprising the respective amino acid sequences of asecond antibody selected from those in Table 2 or 4; or (2) a secondantibody set comprising: (a) a third antibody or antigen-bindingfragment thereof comprising a heavy chain variable region and a lightchain variable region comprising the respective amino acid sequences ofantibody selected from those in Table 2 or 4; and (b) a fourth antibodyor antigen-binding fragment thereof comprising a heavy chain variableregion and a light chain variable region comprising the respective aminoacid sequences of an antibody selected from those in Table 2 or 4,wherein the third antibody is different from the fourth antibody.

In some embodiments, the pharmaceutical composition further comprises asecond therapeutic agent. In some embodiments, the second therapeuticagent comprises an anti-inflammatory drug or an antiviral compound. Insome embodiments, the antiviral compound comprises: a nucleoside analog,a peptoid, an oligopeptide, a polypeptide, a protease inhibitor, a3C-like protease inhibitor, a papain-like protease inhibitor, or aninhibitor of an RNA dependent RNA polymerase. In some embodiments, theantiviral compound may include: acyclovir, gancyclovir, vidarabine,foscarnet, cidofovir, amantadine, ribavirin, trifluorothymidine,zidovudine, didanosine, zalcitabine, or an interferon. In someembodiments, the interferon is an interferon-α or an interferon-β.

Also within the scope of this disclosure is use of the pharmaceuticalcomposition, as described above, in the preparation of a medicament forthe diagnosis, prophylaxis, treatment, or combination thereof of acondition resulting from a SARS-CoV-2.

In another aspect, this disclosure also provides (i) a nucleic acidmolecule encoding a polypeptide chain of the antibody or antigen-bindingfragment thereof described above; (ii) a vector comprising the nucleicacid molecule as described; and (iii) a cultured host cell comprisingthe vector as described.

Also provided is a method for producing a polypeptide, comprising: (a)obtaining the cultured host cell as described; (b) culturing thecultured host cell in a medium under conditions permitting expression ofa polypeptide encoded by the vector and assembling of an antibody orfragment thereof; and (c) purifying the antibody or fragment from thecultured cell or the medium of the cell.

In another aspect, this disclosure provides a kit comprising apharmaceutically acceptable dose unit of the antibody or antigen-bindingfragment thereof or the pharmaceutical composition as described above.Also within the scope of this disclosure is a kit for the diagnosis,prognosis, or monitoring of the treatment of SARS-CoV-2 in a subject,comprising: the antibody or antigen-binding fragment thereof asdescribed; and a least one detection reagent that binds specifically tothe antibody or antigen-binding fragment thereof.

In yet another aspect, this disclosure further provides a method ofneutralizing SARS-CoV-2 in a subject. The method comprises administeringto a subject in need thereof a therapeutically effective amount of theantibody or antigen-binding fragment thereof or a therapeuticallyeffective amount of the pharmaceutical composition, as described herein.

In yet another aspect, this disclosure additionally provides a method ofpreventing or treating a SARS-CoV-2 infection. The method comprisesadministering to a subject in need thereof a therapeutically effectiveamount of the antibody or antigen-binding fragment thereof or atherapeutically effective amount of the pharmaceutical composition, asdescribed herein.

In some embodiments, the method of neutralizing SARS-CoV-2 in a subjectcomprises administering to a subject in need thereof a therapeuticallyeffective amount of a first antibody or antigen-binding fragment thereofand a second antibody or antigen-binding fragment thereof of theantibody or antigen-binding fragment, as described above, wherein thefirst antibody or antigen-binding fragment thereof and the secondantibody or antigen binding fragment thereof exhibit synergisticactivity or a therapeutically effective amount of the pharmaceuticalcomposition described above.

In some embodiments, the method of preventing or treating a SARS-CoV-2infection, comprising administering to a subject in need thereof atherapeutically effective amount of a first antibody or antigen-bindingfragment thereof and a second antibody or antigen-binding fragmentthereof of the antibody or antigen-binding fragment, as described above,wherein the first antibody or antigen-binding fragment thereof and thesecond antibody or antigen binding fragment thereof exhibit synergisticactivity or a therapeutically effective amount of the pharmaceuticalcomposition described above. In some embodiments, the first antibody orantigen-binding fragment thereof is administered before, after, orconcurrently with the second antibody or antigen-binding fragmentthereof.

In some embodiments, the first antibody or antigen-binding fragmentthereof and the second antibody or antigen-binding fragment thereof canbe any combinations of the antibody or antigen-binding fragment thereofcomprising a heavy chain and a light chain that comprise the respectiveamino acid sequences of an antibody selected from those in Table 2 or 4.

In some embodiments, the second therapeutic agent comprises ananti-inflammatory drug or an antiviral compound. In some embodiments,the antiviral compound comprises a nucleoside analog, a peptoid, anoligopeptide, a polypeptide, a protease inhibitor, a 3C-like proteaseinhibitor, a papain-like protease inhibitor, or an inhibitor of an RNAdependent RNA polymerase. In some embodiments, the antiviral compoundmay include: acyclovir, gancyclovir, vidarabine, foscarnet, cidofovir,amantadine, ribavirin, trifluorothymidine, zidovudine, didanosine,zalcitabine, or an interferon. In some embodiments, the interferon is aninterferon-α or an interferon-β.

In some embodiments, the antibody or antigen-binding fragment thereof isadministered before, after, or concurrently with the second therapeuticagent or therapy. In some embodiments, the antibody or antigen-bindingfragment thereof is administered to the subject intravenously,subcutaneously, or intraperitoneally. In some embodiments, the antibodyor antigen-binding fragment thereof is administered prophylactically ortherapeutically.

In another aspect, this disclosure further provides a method fordetecting the presence of SARS CoV-2 in a sample comprising the stepsof: (i) contacting a sample with the antibody or antigen-bindingfragment thereof described above; and (ii) determining binding of theantibody or antigen-binding fragment to one or more SARS CoV-2 antigens,wherein binding of the antibody to the one or more SARS CoV-2 antigensis indicative of the presence of SARS CoV-2 in the sample. In someembodiments, the sample is a blood sample.

In some embodiments, the SARS-CoV-2 antigen comprises a S polypeptide,such as a S polypeptide of a human or an animal SARS-CoV-2. In someembodiments, the SARS-CoV-2 antigen comprises the receptor-bindingdomain (RBD) of the S polypeptide. In some embodiments, the RBDcomprises amino acids 319-541 of the S polypeptide.

In some embodiments, the antibody or antigen-binding fragment thereof isconjugated to a label. In some embodiments, the step of detectingcomprises contacting a secondary antibody with the antibody orantigen-binding fragment thereof and wherein the secondary antibodycomprises a label. In some embodiments, the label includes a fluorescentlabel, a chemiluminescent label, a radiolabel, and an enzyme.

In some embodiments, the step of detecting comprises detectingfluorescence or chemiluminescence. In some embodiments, the step ofdetecting comprises a competitive binding assay or ELISA.

In some embodiments, the method further comprises binding the sample toa solid support. In some embodiments, the solid support includesmicroparticles, microbeads, magnetic beads, and an affinity purificationcolumn.

The foregoing summary is not intended to define every aspect of thedisclosure, and additional aspects are described in other sections, suchas the following detailed description. The entire document is intendedto be related as a unified disclosure, and it should be understood thatall combinations of features described herein are contemplated, even ifthe combination of features are not found together in the same sentence,or paragraph, or section of this document. Other features and advantagesof the invention will become apparent from the following detaileddescription. It should be understood, however, that the detaileddescription and the specific examples, while indicating specificembodiments of the disclosure, are given by way of illustration only,because various changes and modifications within the spirit and scope ofthe disclosure will become apparent to those skilled in the art fromthis detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, and 1i are a set of diagramsshowing plasma neutralizing activity of the neutralizing antibodies.FIG. 1a shows SARS-CoV-2 pseudovirus neutralization assay. NT₅₀ valuesfor COVID-19 convalescent plasma were measured at 1.3 months and 6.2months as well as plasma from mRNA-vaccinees. NT₅₀ values lower than 10were plotted at 10. Mean of 2 independent experiments. Red bars andindicated values represent geometric mean NT₅₀ values. Statisticalsignificance was determined using a two-tailed Mann-Whitney U-test. FIG.1b shows NT₅₀ values for Moderna and Pfizer/BioNTech vaccine recipients.Bars and indicated values represent geometric mean NT₅₀ values.Statistical significance was determined using a two-tailed Mann-WhitneyU-test. FIG. 1c shows anti-RBD IgG AUC (Y axis) plotted against NT₅₀ (Xaxis) r=0.84, p<0.0001. FIG. 1d shows anti-S IgG AUC (Y axis) plottedagainst NT₅₀ (X axis) r=0.83, p<0.0001. FIG. 1e shows anti-RBD IgG AUC(Y axis) plotted against time between the first dose and blood draw (Xaxis) r=−0.60 p=0.0055. FIG. 1f shows anti-S IgG AUC (Y axis) plottedagainst time between the first dose and blood draw (X axis) r=−0.62p=0.0038. FIG. 1g shows NT₅₀ (Y axis) plotted against time between thefirst dose and blood draw (X axis) r=−0.69 p=0.0008. The r and p valuesfor correlations in FIGS. 1c-g were determined by two-tailed Spearman's.FIG. 1h shows examples of neutralization assays, comparing thesensitivity of pseudotyped viruses with WT and RBD mutant SARS-CoV-2 Sproteins to vaccinee plasma. FIG. 1i shows NT₅₀ values for vaccineeplasma (n=15) neutralization of pseudotyped viruses with WT and theindicated RBD-mutant SARS-CoV-2 S proteins. Statistical significance wasdetermined. All experiments were performed a minimum of 2 times.

FIGS. 2a, 2b, 2c, 2d, 2e, 2f, 2g, and 2h are a set of diagrams showingcharacterization of memory B cell antibodies. FIG. 2a showsrepresentative flow cytometry plots showing dual AlexaFluor-647-RBD andPE-RBD binding B cells for 4 vaccinees. FIG. 2b , as in FIG. 2a , showsa dot plot summarizing the percentage of RBD binding B cells in 19vaccinees, in comparison to a cohort of infected individuals assays 1.3and 6.2 months after infection. The horizontal bars indicate meanvalues. Statistical significance was determined using a two-tailedMann-Whitney U-tests. FIG. 2c shows pie charts depicting thedistribution of antibody sequences from the 4 individuals in FIG. 2a .The number in the inner circle indicates the number of sequencesanalyzed. Pie slice size is proportional to the number of clonallyrelated sequences. The black outline indicates the frequency of clonallyexpanded sequences. FIG. 2d , as in FIG. 2c , is a graph showingrelative clonality among 14 vaccinees assayed. Statistical significancewas determined using a two-tailed Mann-Whitney U-tests. FIG. 2e is agraph showing relative abundance of human IGVH genes Sequence ReadArchive accession SRP010970 and vaccinees. A two-sided binomial test wasused to compare the frequency distributions; significant differences aredenoted with stars. FIG. 2f shows clonal relationships between sequencesfrom 14 vaccinated individuals (Moderna and Pfizer in Table 2) andnaturally infected individuals. Interconnecting lines indicate therelationship between antibodies that share V and J gene segmentsequences at both IGH and IGL. FIG. 2g shows number of somaticnucleotide mutations in the IGVH (top) and IGVL (bottom) in vaccineeantibodies (Table 2) compared to natural infection obtained 1.3 or 6.2months after infection. Statistical significance was determined usingthe two-tailed Mann-Whitney U-tests, and the horizontal bars indicatemean values. FIG. 2h , as in FIG. 2g , but for CDR3 length.

FIGS. 3a, 3b, and 3c are a set of diagrams showing anti-SARS-CoV-2 RBDmonoclonal antibody neutralizing activity. FIG. 3a shows the results ofa SARS-CoV-2 pseudovirus neutralization assay. IC₅₀ values forantibodies cloned from COVID-19 convalescent patients were measured at1.3 and 6.2 months as well as antibodies cloned from mRNA-vaccinees.Antibodies with IC₅₀ values above 1000 ng/ml were plotted at 1000 ng/ml.Mean of 2 independent experiments. Red bars and indicated valuesrepresent geometric mean IC₅₀ values in ng/ml. Statistical significancewas determined using a two-tailed Mann-Whitney U-test. FIG. 3b showsIC₅₀ values for 17 selected mAbs for neutralization of wild type and theindicated mutant SARS-CoV-2 pseudoviruses. FIG. 3c shows that antibodyselection pressure can drive emergence of S variants in cell culture;the percentage of sequence reads bearing the indicated RBD mutationsafter a single passage of rVSV/SARS-CoV-2 in the presence of theindicated antibodies is tabulated. Values represent the decimalfrequency with which each mutation occurs as assessed by sequencing.

FIGS. 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i, 4j, 4k , 4 l, 4 m, and 4 n area set of diagrams showing cryo-EM reconstructions of Fab-S complexes.Cryo-EM densities for Fab-S complexes (FIGS. 4a-e ; FIGS. 4k-l ) andclose-up views of antibody footprints on RBDs (FIGS. 4f-j ; FIGS. 4m-n )are shown for neutralizing mAbs. As expected, due to Fab inter-domainflexibility, cryo-EM densities (FIGS. 4a-e ; FIGS. 4k-l ) were weak forthe Fab C_(H)-C_(L) domains. Antibody footprints on RBDs (FIGS. 4f-j ;FIGS. 4m-n ) are presented as Fab V_(H)-V_(L) domains (cartoon)complexed with the RBD (surface). FIGS. 4a and 4f , C669; FIGS. 4b and4g , C643; FIGS. 4c and 4h , C603; FIGS. 4d and 4i , C601; FIGS. 4e and4j , C670; FIGS. 4k and 4m , C666; and FIGS. 4l and 4n , C669. RBDresidues K417, N439, N440, E484, and N501 are highlighted as redsurfaces. The N343 glycan is shown as a teal sphere. FIG. 4o shows acomposite model illustrating targeted epitopes of RBD-specificneutralizing mAbs (shown as V_(H)-V_(L) domains in colors from panelsFIGS. 4a-l ) elicited from mRNA vaccines.

FIGS. 5a, 5b, 5c, 5d, 5e, 5f, 5g, 5h, and 5i are a set of diagramsshowing characterization of plasma antibodies against SARS-CoV-2. FIGS.5a, 5b, 5c, 5d, 5e, and 5f show the results of ELISAs measuring plasmareactivity to S (FIGS. 5a, 5c, and 5e ) and RBD protein (FIGS. 5b, 5d ,and 5f) of 20 vaccinees (grey curves) and 8 controls (black curves).FIG. 5a , Anti-S IgG. FIG. 5b , Anti-RBD IgG. FIG. 5c , Anti-S IgM. FIG.5d , Anti-RBD IgM. FIG. 5e , Anti-S IgA. FIG. 5f , Anti-RBD IgA. Left,optical density at 450 nm (OD 450 nm) for the indicated reciprocalplasma dilutions. Right, normalized area under the curve (AUC) valuesfor the 8 controls and 20 vaccinees. Horizontal bars indicate geometricmean. Statistical significance was determined using the two-tailedMann-Whitney U-test. Average of two or more experiments. FIGS. 5g, 5h,and 5i show correlations of plasma antibodies measurements. FIG. 5gshows normalized AUC for IgG anti-S plotted against normalized AUC forIgG anti-RBD. FIG. 5h shows normalized AUC for IgM anti-S plottedagainst normalized AUC for IgM anti-RBD. FIG. 5i shows normalized AUCfor IgA anti-S plotted against normalized AUC for IgA anti-RBD. The rand p values in FIGS. 5g-i were determined with the two-tailedSpearman's correlation test.

FIGS. 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, and 6j are a set of diagramsshowing plasma neutralizing activity and binding correlations. FIG. 6ashows anti-S IgM AUC (Y axis) plotted against NT₅₀ (X axis) r=0.12,p<0.6179. FIG. 6b shows anti-S IgA AUC (Y axis) plotted against NT₅₀ (Xaxis) r=0.79, p<0.0001. FIG. 6c shows anti-RBD IgM AUC (Y axis) plottedagainst NT50 (X axis) r=−0.05 p=0.8502. FIG. 6d shows anti-RBD IgA AUC(Y axis) plotted against NT50 (X axis) r=0.70 p=0.0006. FIG. 6e showsNT₅₀ (Y axis) plotted against time between last dose and blood draw (Xaxis) r=−0.63 p=0.0032. FIG. 6f shows NT₅₀ (Y axis) plotted against timebetween doses (X axis) r=0.03 p=0.8906. FIG. 6g shows anti-RBD IgG AUC(Y axis) plotted against time between last dose and blood draw (X axis)r=−0.57 p=0.0084. FIG. 6h shows anti-S IgG AUC (Y axis) plotted againsttime between last dose and blood draw (X axis) r=−0.59 p=0.0064. FIG. 6ishows Age (Y axis) plotted against NT₅₀ (X axis) r=−0.06 p=0.8150. The rand p values were determined by two-tailed Spearman's. FIG. 6j showsNT50 values for vaccinee plasma neutralization of pseudotyped viruseswith WT and the indicated RBD-mutant SARS-CoV-2 S proteins.

FIGS. 7a, 7b, 7c, and 7d are a set of diagrams showing the results offlow cytometry. FIG. 7a shows gating strategy used for cell sorting.Gating was on singlets that were CD20+ and CD3−CD8−CD16−OVA−. Sortedcells were RBD-PE+ and RBD-AF647+. FIG. 7b shows the results of flowcytometry depicting the percentage of RBD-double positive memory B cellsfrom a pre-COVID-19 control (HD) and 15 vaccinees. FIG. 7c shows thepercentage of RBD-binding memory B cells in vaccinees (Y axis) plottedagainst time between the first dose and blood draw (X axis) r=0.4028p=0.0873 (left panel), and between last dose and blood draw (X axis)r=0.3319 p=0.1651 (right panel). FIG. 7d shows pie charts depicting thedistribution of antibody sequences from 10 individuals in FIG. 7b . Thenumber in the inner circle indicates the number of sequences analyzed.Pie slice size is proportional to the number of clonally relatedsequences. The black outline indicates the frequency of clonallyexpanded sequences. The r and p values for correlations in FIG. 7c wasdetermined by two-tailed Spearman's.

FIGS. 8a and 8b are a set of diagrams showing frequency distributions ofhuman VL genes. FIG. 8a is a graph showing relative abundance of humanIGVK (left) and IgVL (right) genes of Sequence Read Archive accessionSRP010970 and vaccinees. Two-sided binomial tests with unequal variancewere used to compare the frequency distributions, significantdifferences are denoted with stars. (* p<0.05, ** p<0.01, *** p<0.001,****=p<0.0001). FIG. 8b shows sequences from 14 individuals (Table 2)with clonal relationships depicted as in FIG. 8a . Interconnecting linesindicate the relationship between antibodies that share V and J genesegment sequences at both IGH and IGL.

FIGS. 9a and 9b are a set of diagrams showing antibody somatichypermutation and CDR3 length. FIG. 9a shows number of somaticnucleotide mutations in both the IGVH and IGVL in 14 participants(left). For each individual, the number of the amino acid length of theCDR3s at the IGVH and IGVL is shown (right). The horizontal barsindicate the mean. The number of antibody sequences (IGVH and IGVL)evaluated for each participant are n=68 (MOD 1), n=45 (MOD 2), n=117(MOD 3), n=123 (MOD 4), n=110 (MOD 6), n=109 (MOD 7), n=144 (MOD 8),n=102 (MOD 9), n=132 (PFZ10), n=109 (MOD 11), n=91 (PFZ12), n=78 (C001),n=66 (C003), and n=115 (C004). FIG. 9b shows distribution of thehydrophobicity GRAVY scores at the IGH CDR3 compared to a publicdatabase (see Methods for statistical analysis). The box limits are atthe lower and upper quartiles, the center line indicates the median, thewhiskers are 1.5× interquartile range, and the dots represent outliers.Statistical significance was determined using two-tailed Wilcoxonmatched-pairs signed rank test (n.s.=non-significant, ****=p<0.0001).

FIGS. 10a, 10b, 10c, 10d, 10e, 10f, 10g, 10h, 10i, 10j, 10k and 10 l area set of diagrams showing the results of monoclonal antibody ELISAs.FIG. 10a is a graph showing antibody binding to SARS-CoV-2 RBD. ELISAEC₅₀ (half-maximal response) values for 84 antibodies isolated fromModerna vaccinees measured at 8 weeks after the boost and fromconvalescent individuals at 1.3 and 6.2 months. Horizontal bars indicatea geometric mean. Statistical significance was determined using atwo-tailed Mann-Whitney U-test. Average of two or more experiments.FIGS. 10b-k are graphs showing ELISA titrations for antibodies in FIG.10a . n=84 samples and isotype and control antibodies as indicated inthe figure. FIG. 10l is a table showing a heat map summary of EC₅₀values for binding to wild type RBD and the indicated mutants for 17selected antibodies.

DETAILED DESCRIPTION OF THE INVENTION

SARS-CoV-2 represents a serious public health concern. Methods todiagnose and treat persons who are infected with SARS-CoV-2 provide theopportunity to either prevent or control further spread of infection bySARS-CoV-2. These methods are especially important due to the ability ofSARS-CoV-2 to infect persons through an airborne route.

This invention is based, at least in part, on unexpected neutralizingactivities of the disclosed anti-SARS-CoV-2 antibodies orantigen-binding fragments thereof. These antibodies and antigen-bindingfragments constitute a novel therapeutic strategy in protection fromSARS-CoV-2 infections.

Neutralizing Anti-Sars-Cov-2 Antibodies Antibodies

The invention disclosed herein involves neutralizing anti-SARS-CoV-2antibodies or antigen-binding fragments thereof. These antibodies referto a class of neutralizing antibodies that neutralize multipleSARS-CoV-2 virus strains. The antibodies are able to protect a subjectprophylactically and therapeutically against a lethal challenge with aSARS-CoV-2 virus.

In one aspect, this disclosure provides an isolated anti-SARS-CoV-2antibody or antigen-binding fragment thereof that binds specifically toa SARS-CoV-2 antigen. In some embodiments, the SARS-CoV-2 antigencomprises a portion of a Spike (S) polypeptide, such as a S polypeptideof a human or an animal SARS-CoV-2. In some embodiments, the SARS-CoV-2antigen comprises the receptor-binding domain (RBD) of the Spolypeptide. In some embodiments, the RBD comprises amino acids 319-541of the S polypeptide. In some embodiments, the antibody orantigen-binding fragment thereof is capable of neutralizing a pluralityof SARS-CoV-2 strains.

In some embodiments, the antibody or antigen-binding fragment thereof iscapable of neutralizing a SARS-CoV-2 virus at an IC50 concentration ofless than 50 (e.g., 1, 5, 10, 20, 30, 40, 50) μg/ml.

The spike protein is important because it is present on the outside ofintact SARS-CoV-2. Thus, it presents a target that can be used toinhibit or eliminate an intact virus before the virus has an opportunityto infect a cell. A representative amino acid sequence is providedbelow:

(Accession ID: NC_045512.2) (SEQ ID NO: 357)MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

Listed below in Tables 2 and 4 are amino acid sequences of the heavychain (HC) variable regions and light chain (LC) variable regions ofexemplary antibodies.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises: three heavy chain complementarity determining regions (HCDRs)(HCDR1, HCDR2, and HCDR3) of a heavy chain variable region having theamino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123,125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151,153, 155, 157, 159, 161, 163, 165, or 167; and three light chain CDRs(LCDR1, LCDR2, and LCDR3) of a light chain variable region having theamino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 158, 160, 162, 164, 166, or 168.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises: (i) a heavy chain variable region having an amino acidsequence with at least 75% identity to one selected from those in Table2 or 4; and/or (ii) a light chain variable region having an amino acidsequence with at least 75% identity to one selected from those in Table2 or 4.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises: (i) a heavy chain variable region having the amino acidsequence of one selected from those in Table 2 or 4; and/or (ii) a lightchain variable region having the amino acid sequence of one selectedfrom those in Table 2 or 4.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises a heavy chain variable region and a light chain variableregion that comprises an amino acid sequence pair selected from those inTable 2 or 4.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises: a heavy chain variable region having an amino acid sequencewith at least 75% identity to the amino acid sequence of SEQ ID NO: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, or167; or having the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143,145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, or 167; and alight chain variable region having an amino acid sequence with at least75% identity to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, or 168; orhaving the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166, or 168.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises a heavy chain variable region and a light chain variableregion comprise the respective amino acid sequences of SEQ ID NOs: 1-2,3-4, 5-6, 7-8, 9-10, 11-12, 13-14, 15-16, 17-18, 19-20, 21-22, 23-24,25-26, 27-28, 29-30, 31-32, 33-34, 35-36, 37-38, 39-40, 41-42, 43-44,45-46, 47-48, 49-50, 51-52, 53-54, 55-56, 57-58, 59-60, 61-62, 63-64,65-66, 67-68, 69-70, 71-72, 73-74, 75-76, 77-78, 79-80, 81-82, 83-84,85-86, 87-88, 89-90, 91-92, 93-94, 95-96, 97-98, 99-100, 101-102,103-104, 105-106, 107-108, 109-110, 111-112, 113-114, 115-116, 117-118,119-120, 121-122, 123-124, 125-126, 127-128, 129-130, 131-132, 133-134,135-136, 137-138, 139-140, 141-142, 143-144, 145-146, 147-148, 149-150,151-152, 153-154, 155-156, 157-158, 159-160, 161-162, 163-164, 165-166,or 167-168.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises (a) a first target binding site that specifically binds to anepitope within the S polypeptide, and (b) a second target binding sitethat binds to a different epitope on the S polypeptide or on a differentmolecule. In some embodiments, the multivalent antibody is a bivalent orbispecific antibody.

In some embodiments, the antibody or the antigen-binding fragmentthereof further comprises a variant Fc constant region. In someembodiments, the antibody is a monoclonal antibody. In some embodiments,the antibody is a chimeric antibody, a humanized antibody, or ahumanized monoclonal antibody. In some embodiments, the antibody is asingle-chain antibody, Fab or Fab2 fragment.

In some embodiments, the antibody or the antigen-binding fragmentthereof further comprises a variant Fc constant region. The antibody canbe a monoclonal antibody. In some embodiments, the antibody can be achimeric antibody, a humanized antibody, or a humanized monoclonalantibody. In some embodiments, the antibody can be a single-chainantibody, Fab or Fab2 fragment.

In some embodiments, the antibody or antigen-binding fragment thereofcan be detectably labeled or conjugated to a toxin, a therapeutic agent,a polymer (e.g., polyethylene glycol (PEG)), a receptor, an enzyme, or areceptor ligand. For example, an antibody of the present invention maybe coupled to a toxin (e.g., a tetanus toxin). Such antibodies may beused to treat animals, including humans, that are infected with thevirus that is etiologically linked to SARS-CoV-2. The toxin-coupledantibody is thought to bind to a portion of a spike protein presented onan infected cell, and then kill the infected cell.

In another example, an antibody of the present invention may be coupledto a detectable tag. Such antibodies may be used within diagnosticassays to determine if an animal, such as a human, is infected withSARS-CoV-2. Examples of detectable tags include: fluorescent proteins(i.e., green fluorescent protein, red fluorescent protein, yellowfluorescent protein), fluorescent markers (i.e., fluoresceinisothiocyanate, rhodamine, texas red), radiolabels (i.e., 3H, 32P,1251), enzymes (i.e., β-galactosidase, horseradish peroxidase,β-glucuronidase, alkaline phosphatase), or an affinity tag (i.e.,avidin, biotin, streptavidin). Methods to couple antibodies to adetectable tag are known in the art. Harlow et al., Antibodies: ALaboratory Manual, page 319 (Cold Spring Harbor Pub. 1988).

Fragment

In some embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)2, Fv, and single-chain Fv (scFv) fragments, and otherfragments described below, e.g., diabodies, triabodies tetrabodies, andsingle-domain antibodies. For a review of certain antibody fragments,see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFvfragments, see, e.g., Pluckthun, in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, NewYork), pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos.5,571,894 and 5,587,458. For discussion of Fab and F(ab′)2 fragmentscomprising salvage receptor binding epitope residues and havingincreased in vivo half-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In some embodiments, asingle-domain antibody is a human single-domain antibody (DOMANTIS,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g., E. coli or phage), asdescribed herein.

Chimeric and Humanized Antibodies

In some embodiments, an antibody provided herein is a chimeric antibody.Certain chimeric antibodies are described, e.g., in U.S. Pat. No.4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855(1984)). In one example, a chimeric antibody comprises a non-humanvariable region (e.g., a variable region derived from a mouse, rat,hamster, rabbit, or non-human primate, such as a monkey) and a humanconstant region. In a further example, a chimeric antibody is a “classswitched” antibody in which the class or subclass has been changed fromthat of the parent antibody. Chimeric antibodies include antigen-bindingfragments thereof.

In some embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing specificity determining region(SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing“FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimkaet al., Br. J. Cancer, 83:252-260 (2000) (describing the “guidedselection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

Human Antibodies

In some embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart or using techniques described herein. Human antibodies are describedgenerally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSEtechnology; U.S. Pat. No. 5,770,429 describing HUMAB technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSEtechnology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically displays antibody fragments, eitheras scFv fragments or as Fab fragments. Libraries from immunized sourcesprovide high-affinity antibodies to the immunogen without therequirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned (e.g., from human) to provide a single sourceof antibodies to a wide range of non-self and also self-antigens withoutany immunization as described by Griffiths et al., EMBO J, 12: 725-734(1993). Finally, naive libraries can also be made synthetically bycloning unrearranged V-gene segments from stem cells and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample, U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360. Antibodies or antibodyfragments isolated from human antibody libraries are considered humanantibodies or human antibody fragments herein.

Variants

In some embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen binding.

Substitution, Insertion, and Deletion Variants

In some embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions aredefined herein. Amino acid substitutions may be introduced into anantibody of interest and the products screened for a desired activity,e.g., retained/improved antigen binding, decreased immunogenicity, orimproved antibody-dependent cell-mediated cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC).

Accordingly, an antibody of the invention can comprise one or moreconservative modifications of the CDRs, heavy chain variable region, orlight variable regions described herein. A conservative modification orfunctional equivalent of a peptide, polypeptide, or protein disclosed inthis invention refers to a polypeptide derivative of the peptide,polypeptide, or protein, e.g., a protein having one or more pointmutations, insertions, deletions, truncations, a fusion protein, or acombination thereof. It substantially retains the activity to of theparent peptide, polypeptide, or protein (such as those disclosed in thisinvention). In general, a conservative modification or functionalequivalent is at least 60% (e.g., any number between 60% and 100%,inclusive, e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and99%) identical to a parent. Accordingly, within the scope of thisinvention are heavy chain variable region or light variable regionshaving one or more point mutations, insertions, deletions, truncations,a fusion protein, or a combination thereof, as well as antibodies havingthe variant regions.

As used herein, the percent homology between two amino acid sequences isequivalent to the percent identity between the two sequences. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the presentinvention can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify related sequences.Such searches can be performed using the XBLAST program (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to the antibody molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. (See www.ncbi.nlm.nih.gov).

As used herein, the term “conservative modifications” refers to aminoacid modifications that do not significantly affect or alter the bindingcharacteristics of the antibody containing the amino acid sequence. Suchconservative modifications include amino acid substitutions, additionsand deletions. Modifications can be introduced into an antibody of theinvention by standard techniques known in the art, such as site-directedmutagenesis and PCR-mediated mutagenesis. Conservative amino acidsubstitutions are ones in which the amino acid residue is replaced withan amino acid residue having a similar side chain. Families of aminoacid residues having similar side chains have been defined in the art.These families include: (i) amino acids with basic side chains (e.g.,lysine, arginine, histidine), (ii) acidic side chains (e.g., asparticacid, glutamic acid), (iii) uncharged polar side chains (e.g., glycine,asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), (iv) nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), (v) beta-branched sidechains (e.g., threonine, valine, isoleucine), and (vi) aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

An exemplary substitutional variant is an affinity matured antibody,which may be conveniently generated, e.g., using phage display-basedaffinity maturation techniques such as those described in, e.g.,Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., (2001). Amino acid sequenceinsertions include amino- and/or carboxyl-terminal fusions ranging inlength from one residue to polypeptides containing a hundred or moreresidues, as well as intrasequence insertions of single or multipleamino acid residues. Examples of terminal insertions include an antibodywith an N-terminal methionyl residue. Other insertional variants of theantibody molecule include the fusion to the N- or C-terminus of theantibody to an enzyme (e.g., for ADEPT) or a polypeptide which increasesthe serum half-life of the antibody.

Glycosylation Variants

In some embodiments, an antibody provided herein is altered to increaseor decrease the extent to which the antibody is glycosylated. Additionor deletion of glycosylation sites to an antibody may be convenientlyaccomplished by altering the amino acid sequence such that one or moreglycosylation sites are created or removed.

For example, an aglycoslated antibody can be made (i.e., the antibodylacks glycosylation). Glycosylation can be altered to, for example,increase the affinity of the antibody for antigen. Such carbohydratemodifications can be accomplished by, for example, altering one or moresites of glycosylation within the antibody sequence. For example, one ormore amino acid substitutions can be made that result in elimination ofone or more variable region framework glycosylation sites to therebyeliminate glycosylation at that site. Such aglycosylation may increasethe affinity of the antibody for antigen. Such an approach is describedin further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co et al.

Glycosylation of the constant region on N297 may be prevented bymutating the N297 residue to another residue, e.g., N297A, and/or bymutating an adjacent amino acid, e.g., 298 to thereby reduceglycosylation on N297.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies described herein to thereby produce an antibodywith altered glycosylation. For example, EP 1,176,195 by Hanai et al.describes a cell line with a functionally disrupted FUT8 gene, whichencodes a fucosyltransferase, such that antibodies expressed in such acell line exhibit hypofucosylation. PCT Publication WO 03/035835 byPresta describes a variant Chinese Hamster Ovary cell line, Led 3 cells,with reduced ability to attach fucose to Asn(297)-linked carbohydrates,also resulting in hypofucosylation of antibodies expressed in that hostcell (see also Shields, R. L. et al. (2002) J. Biol. Chem.277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describescell lines engineered to express glycoprotein-modifyingglycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferaseIII (GnTIII)) such that antibodies expressed in the engineered celllines exhibit increased bisecting GlcNac structures which result inincreased ADCC activity of the antibodies (see also Umana et al. (1999)Nat. Biotech. 17: 176-180).

Fc Region Variants

The variable regions of the antibody described herein can be linked(e.g., covalently linked or fused) to an Fc, e.g., an IgG1, IgG2, IgG3or IgG4 Fc, which may be of any allotype or isoallotype, e.g., for IgG1:G1m, G1m1(a), G1m2(x), G1m3(f), G1m17(z); for IgG2: G2m, G2m23(n); forIgG3: G3m, G3m21(g1), G3m28(g5), G3m1 1(b0), G3m5(b1), G3m13(b3),G3m14(b4), G3m10(b5), G3m15(s), G3m16(t), G3m6(c3), G3m24(c5), G3m26(u),G3m27(v); and for K: Km, Km1, Km2, Km3 (see, e.g., Jefferies et al.(2009) mAbs 1:1). In some embodiments, the antibodies variable regionsdescribed herein are linked to an Fc that binds to one or moreactivating Fc receptors (FcγI, FcγIIa or FcγIIIa), and thereby stimulateADCC and may cause T cell depletion. In some embodiments, the antibodyvariable regions described herein are linked to an Fc that causesdepletion.

In some embodiments, the antibody variable regions described herein maybe linked to an Fc comprising one or more modifications, typically toalter one or more functional properties of the antibody, such as serumhalf-life, complement fixation, Fc receptor binding, and/orantigen-dependent cellular cytotoxicity. Furthermore, an antibodydescribed herein may be chemically modified (e.g., one or more chemicalmoieties can be attached to the antibody) or be modified to alter itsglycosylation, to alter one or more functional properties of theantibody. The numbering of residues in the Fc region is that of the EUindex of Kabat.

The Fc region encompasses domains derived from the constant region of animmunoglobulin, preferably a human immunoglobulin, including a fragment,analog, variant, mutant or derivative of the constant region. Suitableimmunoglobulins include IgG1, IgG2, IgG3, IgG4, and other classes suchas IgA, IgD, IgE and IgM. The constant region of an immunoglobulin isdefined as a naturally-occurring or synthetically-produced polypeptidehomologous to the immunoglobulin C-terminal region, and can include aCH1 domain, a hinge, a CH2 domain, a CH3 domain, or a CH4 domain,separately or in combination. In some embodiments, an antibody of thisinvention has an Fc region other than that of a wild type IgA1. Theantibody can have an Fc region from that of IgG (e.g., IgG1, IgG2, IgG3,and IgG4) or other classes such as IgA2, IgD, IgE, and IgM. The Fc canbe a mutant form of IgA1.

The constant region of an immunoglobulin is responsible for manyimportant antibody functions, including Fc receptor (FcR) binding andcomplement fixation. There are five major classes of heavy chainconstant region, classified as IgA, IgG, IgD, IgE, IgM, each withcharacteristic effector functions designated by isotype. For example,IgG is separated into four subclasses known as IgG1, IgG2, IgG3, andIgG4.

Ig molecules interact with multiple classes of cellular receptors. Forexample, IgG molecules interact with three classes of Fcγ receptors(FcγR) specific for the IgG class of antibody, namely FcγRI, FcγRII, andFcγRIIL The important sequences for the binding of IgG to the FcγRreceptors have been reported to be located in the CH2 and CH3 domains.The serum half-life of an antibody is influenced by the ability of thatantibody to bind to an FcR.

In some embodiments, the Fc region is a variant Fc region, e.g., an Fcsequence that has been modified (e.g., by amino acid substitution,deletion and/or insertion) relative to a parent Fc sequence (e.g., anunmodified Fc polypeptide that is subsequently modified to generate avariant), to provide desirable structural features and/or biologicalactivity. For example, one may make modifications in the Fc region inorder to generate an Fc variant that (a) has increased or decreasedADCC, (b) increased or decreased CDC, (c) has increased or decreasedaffinity for C1q and/or (d) has increased or decreased affinity for anFc receptor relative to the parent Fc. Such Fc region variants willgenerally comprise at least one amino acid modification in the Fcregion. Combining amino acid modifications is thought to be particularlydesirable. For example, the variant Fc region may include two, three,four, five, etc. substitutions therein, e.g., of the specific Fc regionpositions identified herein.

A variant Fc region may also comprise a sequence alteration whereinamino acids involved in disulfide bond formation are removed or replacedwith other amino acids. Such removal may avoid reaction with othercysteine-containing proteins present in the host cell used to producethe antibodies described herein. Even when cysteine residues areremoved, single chain Fc domains can still form a dimeric Fc domain thatis held together non-covalently. In other embodiments, the Fc region maybe modified to make it more compatible with a selected host cell. Forexample, one may remove the PA sequence near the N-terminus of a typicalnative Fc region, which may be recognized by a digestive enzyme in E.coli such as proline iminopeptidase. In other embodiments, one or moreglycosylation sites within the Fc domain may be removed. Residues thatare typically glycosylated (e.g., asparagine) may confer cytolyticresponse. Such residues may be deleted or substituted withunglycosylated residues (e.g., alanine). In other embodiments, sitesinvolved in interaction with complement, such as the C1q binding site,may be removed from the Fc region. For example, one may delete orsubstitute the EKK sequence of human IgG1. In some embodiments, sitesthat affect binding to Fc receptors may be removed, preferably sitesother than salvage receptor binding sites. In other embodiments, an Fcregion may be modified to remove an ADCC site. ADCC sites are known inthe art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) withregard to ADCC sites in IgG1. Specific examples of variant Fc domainsare disclosed, for example, in WO 97/34631 and WO 96/32478.

In one embodiment, the hinge region of Fc is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of Fc is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody. In one embodiment, the Fc hinge region of an antibody ismutated to decrease the biological half-life of the antibody. Morespecifically, one or more amino acid mutations are introduced into theCH2-CH3 domain interface region of the Fc-hinge fragment such that theantibody has impaired Staphylococcal protein A (SpA) binding relative tonative Fc-hinge domain SpA binding. This approach is described infurther detail in U.S. Pat. No. 6,165,745 by Ward et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody. For example, one or more aminoacids selected from amino acid residues 234, 235, 236, 237, 297, 318,320 and 322 can be replaced with a different amino acid residue suchthat the antibody has an altered affinity for an effector ligand butretains the antigen-binding ability of the parent antibody. The effectorligand to which affinity is altered can be, for example, an Fc receptoror the CI component of complement. This approach is described in furtherdetail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered C1q binding and/or reduced orabolished CDC. This approach is described in further detail in U.S. Pat.Nos. 6,194,551 by Idusogie et al.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351 by Bodmer et al.

In yet another example, the Fc region may be modified to increase ADCCand/or to increase the affinity for an Fcγ receptor by modifying one ormore amino acids at the following positions: 234, 235, 236, 238, 239,240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262,263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286,289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309,312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333,334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414,416, 419, 430, 433, 434, 435, 436, 437, 438 or 439. Exemplarysubstitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T,332D, and 332E. Exemplary variants include 239D/332E, 236A/332E,236A/239D/332E, 268F/324T, 267E/268F, 267E/324T, and 267E/268F7324T.Other modifications for enhancing FcγR and complement interactionsinclude but are not limited to substitutions 298A, 333A, 334A, 326A,247I, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 305I,and 396L. These and other modifications are reviewed in Strohl, 2009,Current Opinion in Biotechnology 20:685-691.

Fc modifications that increase binding to an Fcγ receptor include aminoacid modifications at any one or more of amino acid positions 238, 239,248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279,280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303,305, 307, 312, 315, 324, 327, 329, 330, 335, 337, 3338, 340, 360, 373,376, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or439 of the Fc region, wherein the numbering of the residues in the Fcregion is that of the EU index as in abat (WO00/42072).

Other Fc modifications that can be made to Fcs are those for reducing orablating binding to FcγR and/or complement proteins, thereby reducing orablating Fc-mediated effector functions such as ADCC, antibody-dependentcellular phagocytosis (ADCP), and CDC. Exemplary modifications includebut are not limited substitutions, insertions, and deletions atpositions 234, 235, 236, 237, 267, 269, 325, and 328, wherein numberingis according to the EU index. Exemplary substitutions include but arenot limited to 234G, 235G, 236R, 237K, 267R, 269R, 325L, and 328R,wherein numbering is according to the EU index. An Fc variant maycomprise 236R/328R. Other modifications for reducing FcγR and complementinteractions include substitutions 297A, 234A, 235A, 237A, 318A, 228P,236E, 268Q, 309L, 330S, 3315, 220S, 226S, 229S, 238S, 233P, and 234V, aswell as removal of the glycosylation at position 297 by mutational orenzymatic means or by production in organisms such as bacteria that donot glycosylate proteins. These and other modifications are reviewed inStrohl, 2009, Current Opinion in Biotechnology 20:685-691.

Optionally, the Fc region may comprise a non-naturally occurring aminoacid residue at additional and/or alternative positions known to oneskilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375;6,737,056; 6,194,551; 7,317,091; 8,101,720; WO00/42072; WO01/58957;WO02/06919; WO04/016750; WO04/029207; WO04/035752; WO04/074455;WO04/099249; WO04/063351; WO05/070963; WO05/040217, WO05/092925 andWO06/020114).

Fc variants that enhance affinity for an inhibitory receptor FcγRIIb mayalso be used. Such variants may provide an Fc fusion protein withimmune-modulatory activities related to FcγRIIb cells, including, forexample, B cells and monocytes. In one embodiment, the Fc variantsprovide selectively enhanced affinity to FcγRIIb relative to one or moreactivating receptors. Modifications for altering binding to FcγRIIbinclude one or more modifications at a position selected from the groupconsisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327,328, and 332, according to the EU index. Exemplary substitutions forenhancing FcγRIIb affinity include but are not limited to 234D, 234E,234F, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E,266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E.Exemplary substitutions include 235Y, 236D, 239D, 266M, 267E, 268D,268E, 328F, 328W, and 328Y. Other Fc variants for enhancing binding toFcγRllb include 235Y/267E, 236D/267E, 239D/268D, 239D/267E, 267E/268D,267E/268E, and 267E/328F.

The affinities and binding properties of an Fc region for its ligand maybe determined by a variety of in vitro assay methods (biochemical orimmunological based assays) known in the art including but not limitedto, equilibrium methods (e.g., ELISA, or radioimmunoassay), or kinetics(e.g., BIACORE analysis), and other methods such as indirect bindingassays, competitive inhibition assays, fluorescence resonance energytransfer (FRET), gel electrophoresis and chromatography (e.g., gelfiltration). These and other methods may utilize a label on one or moreof the components being examined and/or employ a variety of detectionmethods including but not limited to chromogenic, fluorescent,luminescent, or isotopic labels. A detailed description of bindingaffinities and kinetics can be found in Paul, W. E., ed., FundamentalImmunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), whichfocuses on antibody-immunogen interactions.

In some embodiments, the antibody is modified to increase its biologicalhalf-life. Various approaches are possible. For example, this may bedone by increasing the binding affinity of the Fc region for FcRn. Forexample, one or more of the following residues can be mutated: 252, 254,256, 433, 435, 436, as described in U.S. Pat. No. 6,277,375. Specificexemplary substitutions include one or more of the following: T252L,T254S, and/or T256F. Alternatively, to increase the biologicalhalf-life, the antibody can be altered within the CH1 or CL region tocontain a salvage receptor binding epitope taken from two loops of a CH2domain of an Fc region of an IgG, as described in U.S. Pat. Nos.5,869,046 and 6,121,022 by Presta et al. Other exemplary variants thatincrease binding to FcRn and/or improve pharmacokinetic propertiesinclude substitutions at positions 259, 308, 428, and 434, including forexample 259I, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M. Othervariants that increase Fc binding to FcRn include: 250E, 250Q, 428L,428F, 250Q/428L (Hinton et al, 2004, J. Biol. Chem. 279(8): 6213-6216,Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 286A,305A, 307A, 307Q, 311A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields etal, Journal of Biological Chemistry, 2001, 276(9):6591-6604), 252F,252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 311S,433R, 433S, 433I, 433P, 433Q, 434H, 434F, 434Y, 252Y/254T/256E,433K/434F/436H, 308T/309P/311S (Dall Acqua et al. Journal of Immunology,2002, 169:5171-5180, Dall'Acqua et al., 2006, Journal of BiologicalChemistry 281:23514-23524). Other modifications for modulating FcRnbinding are described in Yeung et al., 2010, J Immunol, 182:7663-7671.In some embodiments, hybrid IgG isotypes with particular biologicalcharacteristics may be used. For example, an IgG1/IgG3 hybrid variantmay be constructed by substituting IgG 1 positions in the CH2 and/or CH3region with the amino acids from IgG3 at positions where the twoisotypes differ. Thus a hybrid variant IgG antibody may be constructedthat comprises one or more substitutions, e.g., 274Q, 276K, 300F, 339T,356E, 358M, 384S, 392N, 397M, 422I, 435R, and 436F. In other embodimentsdescribed herein, an IgG1/IgG2 hybrid variant may be constructed bysubstituting IgG2 positions in the CH2 and/or CH3 region with aminoacids from IgG1 at positions where the two isotypes differ. Thus ahybrid variant IgG antibody may be constructed chat comprises one ormore substitutions, e.g., one or more of the following amino acidsubstitutions: 233E, 234L, 235L, 236G (referring to an insertion of aglycine at position 236), and 321 h.

Moreover, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII,and FcRn have been mapped and variants with improved binding have beendescribed (see Shields, R. L. et al. (2001) J. Biol. Chem.276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334,and 339 were shown to improve binding to FcγRIII Additionally, thefollowing combination mutants were shown to improve FcγRIII binding:T256A/S298A, S298A/E333A, S298A/K224A, and S298A/E333A/K334A, which hasbeen shown to exhibit enhanced FcγRIIIa binding and ADCC activity(Shields et al., 2001). Other IgG1 variants with strongly enhancedbinding to FcγRIIIa have been identified, including variants withS239D/I332E and S239D/I332E/A330L mutations which showed the greatestincrease in affinity for FcγRIIIa, a decrease in FcγRIIb binding, andstrong cytotoxic activity in cynomolgus monkeys (Lazar et al. , 2006).Introduction of the triple mutations into antibodies such as alemtuzumab(CD52− specific), trastuzumab (HER2/neu− specific), rituximab (CD20−specific), and cetuximab (EGFR− specific) translated into greatlyenhanced ADCC activity in vitro, and the S239D/I332E variant showed anenhanced capacity to deplete B cells in monkeys (Lazar et al., 2006). Inaddition, IgG1 mutants containing L235V, F243L, R292P, Y300L and P396Lmutations which exhibited enhanced binding to FcγRIIIa and concomitantlyenhanced ADCC activity in transgenic mice expressing human FcγRIIIa inmodels of B cell malignancies and breast cancer have been identified(Stavenhagen et al., 2007; Nordstrom et al., 2011). Other Fc mutantsthat may be used include: S298A/E333A/L334A, S239D/I332E,S239D/I332E/A330L, L235V/F243L/R292P/Y300L/is P396L, and M428L/N434S.

In some embodiments, an Fc is chosen that has reduced binding to FcγRs.An exemplary Fc, e.g., IgG1 Fc, with reduced FcγR binding, comprises thefollowing three amino acid substitutions: L234A, L235E, and G237A.

In some embodiments, an Fc is chosen that has reduced complementfixation. An exemplary Fc, e.g., IgG1 Fc, with reduced complementfixation, has the following two amino acid substitutions: A330S andP331S.

In some embodiments, an Fc is chosen that has essentially no effectorfunction, i.e., it has reduced binding to FcγRs and reduced complementfixation. An exemplary Fc, e.g., IgG1 Fc, that is effectorless,comprises the following five mutations: L234A, L235E, G237A, A330S, andP331S.

When using an IgG4 constant domain, it is usually preferable to includethe substitution S228P, which mimics the hinge sequence in IgG1 andthereby stabilizes IgG4 molecules.

Multivalent Antibodies

In one embodiment, the antibodies of the invention may be monovalent ormultivalent (e.g., bivalent, trivalent, etc.). As used herein, the term“valency” refers to the number of potential target binding sitesassociated with an antibody. Each target binding site specifically bindsone target molecule or specific position or locus on a target molecule.When an antibody is monovalent, each binding site of the molecule willspecifically bind to a single antigen position or epitope. When anantibody comprises more than one target binding site (multivalent), eachtarget binding site may specifically bind the same or differentmolecules (e.g., may bind to different ligands or different antigens, ordifferent epitopes or positions on the same antigen). See, for example,U.S.P.N. 2009/0130105. In each case, at least one of the binding siteswill comprise an epitope, motif or domain associated with a DLL3isoform.

In one embodiment, the antibodies are bispecific antibodies in which thetwo chains have different specificities, as described in Millstein etal., 1983, Nature, 305:537-539. Other embodiments include antibodieswith additional specificities, such as trispecific antibodies. Othermore sophisticated compatible multispecific constructs and methods oftheir fabrication are set forth in U.S.P.N. 2009/0155255, as well as WO94/04690; Suresh et al., 1986, Methods in Enzymology, 121:210; andWO96/27011.

As stated above, multivalent antibodies may immunospecifically bind todifferent epitopes of the desired target molecule or mayimmunospecifically bind to both the target molecule as well as aheterologous epitope, such as a heterologous polypeptide or solidsupport material. In some embodiments, the multivalent antibodies mayinclude bispecific antibodies or trispecific antibodies. Bi specificantibodies also include cross-linked or “heteroconjugate” antibodies.For example, one of the antibodies in the heteroconjugate can be coupledto avidin, the other to biotin. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treatment of HIV infection (WO 91/00360, WO92/200373, and EP 03089). Heteroconjugate antibodies may be made usingany convenient cross-linking methods. Suitable cross-linking agents arewell known in the art and are disclosed in U.S. Pat. No. 4,676,980,along with a number of cross-linking techniques.

In some embodiments, antibody variable domains with the desired bindingspecificities (antibody-antigen combining sites) are fused toimmunoglobulin constant domain sequences, such as an immunoglobulinheavy chain constant domain comprising at least part of the hinge, CH2,and/or CH3 regions, using methods well known to those of ordinary skillin the art.

Antibody Derivatives

An antibody provided herein may be further modified to containadditional nonproteinaceous moieties that are known in the art andreadily available. The moieties suitable for derivatization of theantibody include but are not limited to water-soluble polymers.

Non-limiting examples of water-soluble polymers include, but are notlimited to, PEG, copolymers of ethylene glycol/propylene glycol,carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, polypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymer is attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

Another modification of the antibodies described herein is pegylation.An antibody can be pegylated to, for example, increase the biological(e.g., serum) half-life of the antibody. To pegylate an antibody, theantibody, or fragment thereof, typically is reacted with PEG, such as areactive ester or aldehyde derivative of PEG, under conditions in whichone or more PEG groups become attached to the antibody or antibodyfragment. Preferably, the pegylation is carried out via an acylationreaction or an alkylation reaction with a reactive PEG molecule (or ananalogous reactive water-soluble polymer). As used herein, the term“polyethylene glycol” is intended to encompass any of the forms of PEGthat have been used to derivatize other proteins, such as mono (CI-CIO)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.In some embodiments, the antibody to be pegylated is an aglycosylatedantibody. Methods for pegylating proteins are known in the art and canbe applied to the antibodies described herein. See, for example, EP 0154 316 by Nishimura et al. and EP0401384 by Ishikawa et al.

The present invention also encompasses a human monoclonal antibodydescribed herein conjugated to a therapeutic agent, a polymer, adetectable label or enzyme. In one embodiment, the therapeutic agent isa cytotoxic agent. In one embodiment, the polymer is PEG.

Nucleic Acids, Expression Cassettes, and Vectors

The present invention provides isolated nucleic acid segments thatencode the polypeptides, peptide fragments, and coupled proteins of theinvention. The nucleic acid segments of the invention also includesegments that encode for the same amino acids due to the degeneracy ofthe genetic code. For example, the amino acid threonine is encoded byACU, ACC, ACA, and ACG and is therefore degenerate. It is intended thatthe invention includes all variations of the polynucleotide segmentsthat encode for the same amino acids. Such mutations are known in theart (Watson et al., Molecular Biology of the Gene, Benjamin Cummings1987). Mutations also include alteration of a nucleic acid segment toencode for conservative amino acid changes, for example, thesubstitution of leucine for isoleucine and so forth. Such mutations arealso known in the art. Thus, the genes and nucleotide sequences of theinvention include both the naturally occurring sequences as well asmutant forms.

The nucleic acid segments of the invention may be contained within avector. A vector may include, but is not limited to, any plasmid,phagemid, F-factor, virus, cosmid, or phage in a double- orsingle-stranded linear or circular form which may or may not be selftransmissible or mobilizable. The vector can also transform aprokaryotic or eukaryotic host either by integration into the cellulargenome or exist extra-chromosomally (e.g., autonomous replicatingplasmid with an origin of replication).

Preferably the nucleic acid segment in the vector is under the controlof, and operably linked to, an appropriate promoter or other regulatoryelements for transcription in vitro or in a host cell, such as aeukaryotic cell, or a microbe, e.g., bacteria. The vector may be ashuttle vector that functions in multiple hosts. The vector may also bea cloning vector that typically contains one or a small number ofrestriction endonuclease recognition sites at which foreign DNAsequences can be inserted in a determinable fashion. Such insertion canoccur without loss of essential biological function of the cloningvector. A cloning vector may also contain a marker gene that is suitablefor use in the identification and selection of cells transformed withthe cloning vector. Examples of marker genes are tetracycline resistanceor ampicillin resistance. Many cloning vectors are commerciallyavailable (Stratagene, New England Biolabs, Clonetech).

The nucleic acid segments of the invention may also be inserted into anexpression vector. Typically an expression vector contains prokaryoticDNA elements coding for a bacterial replication origin and an antibioticresistance gene to provide for the amplification and selection of theexpression vector in a bacterial host; regulatory elements that controlinitiation of transcription such as a promoter; and DNA elements thatcontrol the processing of transcripts such as introns, or atranscription termination/polyadenylation sequence.

Methods to introduce nucleic acid segment into a vector are available inthe art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rdedition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001)).Briefly, a vector into which a nucleic acid segment is to be inserted istreated with one or more restriction enzymes (restriction endonuclease)to produce a linearized vector having a blunt end, a “sticky” end with a5′ or a 3′ overhang, or any combination of the above. The vector mayalso be treated with a restriction enzyme and subsequently treated withanother modifying enzyme, such as a polymerase, an exonuclease, aphosphatase or a kinase, to create a linearized vector that hascharacteristics useful for ligation of a nucleic acid segment into thevector. The nucleic acid segment that is to be inserted into the vectoris treated with one or more restriction enzymes to create a linearizedsegment having a blunt end, a “sticky” end with a 5′ or a 3′ overhang,or any combination of the above. The nucleic acid segment may also betreated with a restriction enzyme and subsequently treated with anotherDNA modifying enzyme. Such DNA modifying enzymes include, but are notlimited to, polymerase, exonuclease, phosphatase or a kinase, to createa nucleic acid segment that has characteristics useful for ligation of anucleic acid segment into the vector.

The treated vector and nucleic acid segment are then ligated together toform a construct containing a nucleic acid segment according to methodsavailable in the art (Sambrook et al., Molecular Cloning: A LaboratoryManual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(2001)). Briefly, the treated nucleic acid fragment, and the treatedvector are combined in the presence of a suitable buffer and ligase. Themixture is then incubated under appropriate conditions to allow theligase to ligate the nucleic acid fragment into the vector.

The invention also provides an expression cassette which contains anucleic acid sequence capable of directing expression of a particularnucleic acid segment of the invention, either in vitro or in a hostcell. Also, a nucleic acid segment of the invention may be inserted intothe expression cassette such that an anti-sense message is produced. Theexpression cassette is an isolatable unit such that the expressioncassette may be in linear form and functional for in vitro transcriptionand translation assays. The materials and procedures to conduct theseassays are commercially available from Promega Corp. (Madison, Wis.).For example, an in vitro transcript may be produced by placing a nucleicacid sequence under the control of a T7 promoter and then using T7 RNApolymerase to produce an in vitro transcript. This transcript may thenbe translated in vitro through use of a rabbit reticulocyte lysate.Alternatively, the expression cassette can be incorporated into a vectorallowing for replication and amplification of the expression cassettewithin a host cell or also in vitro transcription and translation of anucleic acid segment.

Such an expression cassette may contain one or a plurality ofrestriction sites allowing for placement of the nucleic acid segmentunder the regulation of a regulatory sequence. The expression cassettecan also contain a termination signal operably linked to the nucleicacid segment as well as regulatory sequences required for propertranslation of the nucleic acid segment. The expression cassettecontaining the nucleic acid segment may be chimeric, meaning that atleast one of its components is heterologous with respect to at least oneof its other components. The expression cassette may also be one that isnaturally occurring but has been obtained in a recombinant form usefulfor heterologous expression. Expression of the nucleic acid segment inthe expression cassette may be under the control of a constitutivepromoter or an inducible promoter, which initiates transcription onlywhen the host cell is exposed to some particular external stimulus.

The expression cassette may include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, anucleic acid segment and a transcriptional and translational terminationregion functional in vivo and/or in vitro. The termination region may benative with the transcriptional initiation region, may be native withthe nucleic acid segment, or may be derived from another source.

The regulatory sequence can be a polynucleotide sequence locatedupstream (5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influences the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences can include, but are not limited to,enhancers, promoters, repressor binding sites, translation leadersequences, introns, and polyadenylation signal sequences. They mayinclude natural and synthetic sequences as well as sequences, which maybe a combination of synthetic and natural sequences. While regulatorysequences are not limited to promoters, some useful regulatory sequencesinclude constitutive promoters, inducible promoters, regulatedpromoters, tissue-specific promoters, viral promoters, and syntheticpromoters.

A promoter is a nucleotide sequence that controls the expression of thecoding sequence by providing the recognition for RNA polymerase andother factors required for proper transcription. A promoter includes aminimal promoter, consisting only of all basal elements needed fortranscription initiation, such as a TATA-box and/or initiator that is ashort DNA sequence comprised of a TATA-box and other sequences thatserve to specify the site of transcription initiation, to whichregulatory elements are added for control of expression. A promoter maybe derived entirely from a native gene, or be composed of differentelements derived from different promoters found in nature, or even becomprised of synthetic DNA segments. A promoter may contain DNAsequences that are involved in the binding of protein factors thatcontrol the effectiveness of transcription initiation in response tophysiological or developmental conditions.

The invention also provides a construct containing a vector and anexpression cassette. The vector may be selected from, but not limitedto, any vector previously described. Into this vector may be inserted anexpression cassette through methods known in the art and previouslydescribed (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rdedition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001)). Inone embodiment, the regulatory sequences of the expression cassette maybe derived from a source other than the vector into which the expressioncassette is inserted. In another embodiment, a construct containing avector and an expression cassette is formed upon insertion of a nucleicacid segment of the invention into a vector that itself containsregulatory sequences. Thus, an expression cassette is formed uponinsertion of the nucleic acid segment into the vector. Vectorscontaining regulatory sequences are available commercially, and methodsfor their use are known in the art (Clonetech, Promega, Stratagene).

In another aspect, this disclosure also provides (i) a nucleic acidmolecule encoding a polypeptide chain of the antibody or antigen-bindingfragment thereof described above; (ii) a vector comprising the nucleicacid molecule as described; and (iii) a cultured host cell comprisingthe vector as described. Also provided is a method for producing apolypeptide, comprising: (a) obtaining the cultured host cell asdescribed; (b) culturing the cultured host cell in a medium underconditions permitting expression of a polypeptide encoded by the vectorand assembling of an antibody or fragment thereof; and (c) purifying theantibody or fragment from the cultured cell or the medium of the cell.

Methods of Production

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment, anisolated nucleic acid encoding an antibody described herein is provided.Such nucleic acid may encode an amino acid sequence comprising the VLand/or an amino acid sequence comprising the VH of the antibody (e.g.,the light and/or heavy chains of the antibody). In a further embodiment,one or more vectors (e.g., expression vectors) comprising such nucleicacid are provided. In a further embodiment, a host cell comprising suchnucleic acid is provided. In one such embodiment, a host cell comprises(e.g., has been transformed with): (1) a vector comprising a nucleicacid that encodes an amino acid sequence comprising the VL of theantibody and an amino acid sequence comprising the VH of the antibody,or (2) a first vector comprising a nucleic acid that encodes an aminoacid sequence comprising the VL of the antibody and a second vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VH of the antibody. In one embodiment, the host cell is eukaryotic,e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0,NS0, Sp20 cell). In one embodiment, a method of making an antibody isprovided, wherein the method comprises culturing a host cell comprisinga nucleic acid encoding the antibody, as provided above, underconditions suitable for expression of the antibody, and optionallyrecovering the antibody from the host cell (or host cell culturemedium).

For recombinant production of an antibody, a nucleic acid encoding anantibody, e.g., as described above, is isolated and inserted into one ormore vectors for further cloning and/or expression in a host cell. Suchnucleic acid may be readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified, which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include CHO cells,including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2/0. Fora review of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol.248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003).

Compositions and Kits

The antibodies of this invention represent an excellent way for thedevelopment of antiviral therapies either alone or in antibody cocktailswith additional anti-SARS-CoV-2 virus antibodies for the treatment ofhuman SARS-CoV-2 infections in humans.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising the antibodies of the present invention describedherein formulated together with a pharmaceutically acceptable carrier.The composition may optionally contain one or more additionalpharmaceutically active ingredients, such as another antibody or atherapeutic agent.

In some embodiments, the pharmaceutical comprises two or more of theantibody or antigen-binding fragment thereof described above, such asany combinations of the antibody or antigen-binding fragment thereofcomprising a heavy chain and a light chain that comprise the respectiveamino acid sequences described herein.

The pharmaceutical compositions of the invention also can beadministered in a combination therapy with, for example, anotherimmune-stimulatory agent, an antiviral agent, or a vaccine, etc. In someembodiments, a composition comprises an antibody of this invention at aconcentration of at least 1 mg/ml, 5 mg/ml, 10 mg/ml, 50 mg/ml, 100mg/ml, 150 mg/ml, 200 mg/ml, 1-300 mg/ml, or 100-300 mg/ml.

In some embodiments, the second therapeutic agent comprises ananti-inflammatory drug or an antiviral compound. In some embodiments,the antiviral compound comprises: a nucleoside analog, a peptoid, anoligopeptide, a polypeptide, a protease inhibitor, a 3C-like proteaseinhibitor, a papain-like protease inhibitor, or an inhibitor of an RNAdependent RNA polymerase. In some embodiments, the antiviral compoundmay include: acyclovir, gancyclovir, vidarabine, foscarnet, cidofovir,amantadine, ribavirin, trifluorothymidine, zidovudine, didanosine,zalcitabine or an interferon. In some embodiments, the interferon is aninterferon-α or an interferon-β.

Also within the scope of this disclosure is use of the pharmaceuticalcomposition in the preparation of a medicament for the diagnosis,prophylaxis, treatment, or combination thereof of a condition resultingfrom a SARS-CoV-2.

The pharmaceutical composition can comprise any number of excipients.Excipients that can be used include carriers, surface-active agents,thickening or emulsifying agents, solid binders, dispersion orsuspension aids, solubilizers, colorants, flavoring agents, coatings,disintegrating agents, lubricants, sweeteners, preservatives, isotonicagents, and combinations thereof. The selection and use of suitableexcipients is taught in Gennaro, ed., Remington: The Science andPractice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), thedisclosure of which is incorporated herein by reference.

Preferably, a pharmaceutical composition is suitable for intravenous,intramuscular, subcutaneous, parenteral, spinal or epidermaladministration (e.g., by injection or infusion). Depending on the routeof administration, the active compound can be coated in a material toprotect it from the action of acids and other natural conditions thatmay inactivate it. The phrase “parenteral administration” as used hereinmeans modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid,intraspinal, epidural and intrasternal injection and infusion.Alternatively, an antibody of the present invention described herein canbe administered via a non-parenteral route, such as a topical, epidermalor mucosal route of administration, e.g., intranasally, orally,vaginally, rectally, sublingually or topically.

The pharmaceutical compositions of the invention may be prepared in manyforms that include tablets, hard or soft gelatin capsules, aqueoussolutions, suspensions, and liposomes and other slow-releaseformulations, such as shaped polymeric gels. An oral dosage form may beformulated such that the antibody is released into the intestine afterpassing through the stomach. Such formulations are described in U.S.Pat. No. 6,306,434 and in the references contained therein.

Oral liquid pharmaceutical compositions may be in the form of, forexample, aqueous or oily suspensions, solutions, emulsions, syrups orelixirs, or may be presented as a dry product for constitution withwater or other suitable vehicle before use. Such liquid pharmaceuticalcompositions may contain conventional additives such as suspendingagents, emulsifying agents, non-aqueous vehicles (which may includeedible oils), or preservatives.

An antibody can be formulated for parenteral administration (e.g., byinjection, for example, bolus injection or continuous infusion) and maybe presented in unit dosage form in ampules, prefilled syringes, smallvolume infusion containers or multi-dose containers with an addedpreservative. The pharmaceutical compositions may take such forms assuspensions, solutions, or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Pharmaceutical compositions suitable for rectaladministration can be prepared as unit dose suppositories. Suitablecarriers include saline solution and other materials commonly used inthe art.

For administration by inhalation, an antibody can be convenientlydelivered from an insufflator, nebulizer or a pressurized pack or otherconvenient means of delivering an aerosol spray. Pressurized packs maycomprise a suitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, anantibody may take the form of a dry powder composition, for example, apowder mix of a modulator and a suitable powder base such as lactose orstarch. The powder composition may be presented in unit dosage form in,for example, capsules or cartridges or, e.g., gelatin or blister packsfrom which the powder may be administered with the aid of an inhalatoror insufflator. For intra-nasal administration, an antibody may beadministered via a liquid spray, such as via a plastic bottle atomizer.

Pharmaceutical compositions of the invention may also contain otheringredients such as flavorings, colorings, anti-microbial agents, orpreservatives. It will be appreciated that the amount of an antibodyrequired for use in treatment will vary not only with the particularcarrier selected but also with the route of administration, the natureof the condition being treated and the age and condition of the patient.Ultimately the attendant health care provider may determine properdosage. In addition, a pharmaceutical composition may be formulated as asingle unit dosage form.

The pharmaceutical composition of the present invention can be in theform of sterile aqueous solutions or dispersions. It can also beformulated in a microemulsion, liposome, or other ordered structuresuitable to high drug concentration.

An antibody of the present invention described herein can beadministered as a sustained release formulation, in which case lessfrequent administration is required. Dosage and frequency vary dependingon the half-life of the antibody in the patient. In general, humanantibodies show the longest half-life, followed by humanized antibodies,chimeric antibodies, and nonhuman antibodies. The dosage and frequencyof administration can vary depending on whether the treatment isprophylactic or therapeutic. In prophylactic applications, a relativelylow dosage is administered at relatively infrequent intervals over along period of time. Some patients continue to receive treatment for therest of their lives. In therapeutic applications, a relatively highdosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferably,until the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patient can be administered a prophylacticregime.

The amount of active ingredient that can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated and the particular mode of administration and willgenerally be that amount of the composition, which produces atherapeutic effect. Generally, out of one hundred percent, this amountwill range from about 0.01% to about 99% of active ingredient,preferably from about 0.1% to about 70%, most preferably from about 1%to about 30% of active ingredient in combination with a pharmaceuticallyacceptable carrier.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus can beadministered, several divided doses can be administered over time or thedose can be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. Alternatively, theantibody can be administered as a sustained release formulation, inwhich case less frequent administration is required. For administrationof the antibody, the dosage ranges from about 0.0001 to 800 mg/kg, andmore usually 0.01 to 5 mg/kg, of the host body weight. For exampledosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg bodyweight, 5 mg/kg body weight or 10 mg/kg body weight or within the rangeof 1-10 mg/kg. An exemplary treatment regime entails administration onceper week, once every two weeks, once every three weeks, once every fourweeks, once a month, once every 3 months or once every three to 6months. Preferred dosage regimens for an antibody of the inventioninclude 1 mg/kg body weight or 3 mg/kg body weight via intravenousadministration, with the antibody being given using one of the followingdosing schedules: (i) every four weeks for six dosages, then every threemonths; (ii) every three weeks; (iii) 3 mg/kg body weight once followedby 1 mg/kg body weight every three weeks. In some methods, dosage isadjusted to achieve a plasma antibody concentration of about 1-1000m/mland in some methods about 25-300 μg/ml. A “therapeutically effectivedosage” of an antibody of the invention preferably results in a decreasein severity of disease symptoms, an increase in frequency and durationof disease symptom-free periods, or a prevention of impairment ordisability due to the disease affliction. For example, for the treatmentof SARS-CoV-2 infection in a subject, a “therapeutically effectivedosage” preferably inhibits SARS-CoV-2 virus replication or uptake byhost cells by at least about 20%, more preferably by at least about 40%,even more preferably by at least about 60%, and still more preferably byat least about 80% relative to untreated subjects. A therapeuticallyeffective amount of a therapeutic compound can neutralize SARS-CoV-2virus, or otherwise ameliorate symptoms in a subject, which is typicallya human or can be another mammal.

The pharmaceutical composition can be a controlled release formulation,including implants, transdermal patches, and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered via medical devices such as(1) needleless hypodermic injection devices (e.g., U.S. Pat. Nos.5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and4,596,556); (2) micro-infusion pumps (U.S. Pat. No. 4,487,603); (3)transdermal devices (U.S. Pat. No. 4,486,194); (4) infusion apparati(U.S. Pat. Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S.Pat. Nos. 4,439,196 and 4,475,196); the disclosures of which areincorporated herein by reference.

In some embodiments, the human monoclonal antibodies of the inventiondescribed herein can be formulated to ensure proper distribution invivo. For example, to ensure that the therapeutic compounds of theinvention cross the blood-brain barrier, they can be formulated inliposomes, which may additionally comprise targeting moieties to enhanceselective transport to specific cells or organs. See, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; 5,416,016; and 5,399,331; V. V. Ranade (1989)Clin. Pharmacol. 29:685; Umezawa et al., (1988) Biochem. Biophys. Res.Commun. 153:1038; Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais etal. (1995) Antimicrob. Agents Chemother. 39:180; Briscoe et al. (1995)Am. Physiol. 1233:134; Schreier et al. (1994). Biol. Chem. 269:9090;Keinanen and Laukkanen (1994) FEBS Lett. 346:123; and Killion and Fidler(1994) Immunomethods 4:273.

In some embodiments, the initial dose may be followed by administrationof a second or a plurality of subsequent doses of the antibody orantigen-binding fragment thereof in an amount that can be approximatelythe same or less than that of the initial dose, wherein the subsequentdoses are separated by at least 1 day to 3 days; at least one week, atleast 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; atleast 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; atleast 10 weeks; at least 12 weeks; or at least 14 weeks.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor-mediated endocytosis (see, e.g.,Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, transdermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The composition may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. The pharmaceutical composition can also be deliveredin a vesicle, in particular, a liposome (see, for example, Langer (1990)Science 249: 1527-1533).

The use of nanoparticles to deliver the antibodies of the presentinvention is also contemplated herein. Antibody-conjugated nanoparticlesmay be used both for therapeutic and diagnostic applications.Antibody-conjugated nanoparticles and methods of preparation and use aredescribed in detail by Arruebo, M., et al. 2009 (“Antibody-conjugatednanoparticles for biomedical applications” in J. Nanomat. Volume 2009,Article ID 439389), incorporated herein by reference. Nanoparticles maybe developed and conjugated to antibodies contained in pharmaceuticalcompositions to target cells. Nanoparticles for drug delivery have alsobeen described in, for example, U.S. Pat. No. 8,257,740, or U.S. Pat.No. 8,246,995, each incorporated herein in its entirety.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used.In another embodiment, polymeric materials can be used. In yet anotherembodiment, a controlled release system can be placed in proximity ofthe composition's target, thus requiring only a fraction of the systemicdose.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous, intracranial, intraperitoneal andintramuscular injections, drip infusions, etc. These injectablepreparations may be prepared by methods publicly known. For example, theinjectable preparations may be prepared, e.g., by dissolving, suspendingor emulsifying the antibody or its salt described above in a sterileaqueous medium or an oily medium conventionally used for injections. Asthe aqueous medium for injections, there are, for example, physiologicalsaline, an isotonic solution containing glucose and other auxiliaryagents, etc., which may be used in combination with an appropriatesolubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol(e.g., propylene glycol, polyethylene glycol), a nonionic surfactant[e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct ofhydrogenated castor oil)], etc. As the oily medium, there are employed,e.g., sesame oil, soybean oil, etc., which may be used in combinationwith a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.The injection thus prepared is preferably filled in an appropriateampoule.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but certainlyare not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™,OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis,Frankfurt, Germany), to name only a few. Examples of disposable pendelivery devices having applications in subcutaneous delivery of apharmaceutical composition of the present invention include, butcertainly are not limited to the SOLOSTAR™ pen (Sanofi-Aventis), theFLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™Autoinjector (Amgen, Thousand Oaks, Calif.), the PENLET™ (Haselmeier,Stuttgart, Germany), the EPIPEN (Dey, L. P.) and the HUMIRA™ Pen (AbbottLabs, Abbott Park, Ill.), to name only a few.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the antibody contained isgenerally about 5 to about 500 mg per dosage form in a unit dose;especially in the form of injection, it is preferred that the antibodyis contained in about 5 to about 300 mg and in about 10 to about 300 mgfor the other dosage forms.

In another aspect, this disclosure provides a kit comprising apharmaceutically acceptable dose unit of the antibody or antigen-bindingfragment thereof of or the pharmaceutical composition as describedabove. Also within the scope of this disclosure is a kit for thediagnosis, prognosis or monitoring of treatment of SARS-CoV-2 in asubject, comprising: the antibody or antigen-binding fragment thereof asdescribed; and a least one detection reagent that binds specifically tothe antibody or antigen-binding fragment thereof.

In some embodiments, the kit also includes a container that contains thecomposition and optionally informational material. The informationalmaterial can be descriptive, instructional, marketing or other materialthat relates to the methods described herein and/or the use of theagents for therapeutic benefit. In an embodiment, the kit also includesan additional therapeutic agent, as described above. For example, thekit includes a first container that contains the composition and asecond container for the additional therapeutic agent.

The informational material of the kits is not limited in its form. Insome embodiments, the informational material can include informationabout production of the composition, concentration, date of expiration,batch or production site information, and so forth. In one embodiment,the informational material relates to methods of administering thecomposition, e.g., in a suitable dose, dosage form, or mode ofadministration (e.g., a dose, dosage form, or mode of administrationdescribed herein), to treat a subject in need thereof. In oneembodiment, the instructions provide a dosing regimen, dosing schedule,and/or route of administration of the composition or the additionaltherapeutic agent. The information can be provided in a variety offormats, including printed text, computer-readable material, videorecording, or audio recording, or information that contains a link oraddress to substantive material.

The kit can include one or more containers for the composition. In someembodiments, the kit contains separate containers, dividers, orcompartments for the composition and informational material. Forexample, the composition can be contained in a bottle or vial, and theinformational material can be contained in a plastic sleeve or packet.In other embodiments, the separate elements of the kit are containedwithin a single, undivided container. For example, the composition iscontained in a bottle or vial that has attached thereto theinformational material in the form of a label. In some embodiments, thekit includes a plurality (e.g., a pack) of individual containers, eachcontaining one or more unit dosage forms (e.g., a dosage form describedherein) of the agents.

The kit optionally includes a device suitable for administration of thecomposition or other suitable delivery device. The device can beprovided pre-loaded with one or both of the agents or can be empty, butsuitable for loading. Such a kit may optionally contain a syringe toallow for injection of the antibody contained within the kit into ananimal, such as a human.

Methods of Use Methods of Treatment

The antibodies, compositions, and formulations described herein can beused to neutralize SARS-CoV-2 virus and thereby treating or preventingSARS-CoV-2 infections.

Accordingly, in one aspect, this disclosure further provides a method ofneutralizing SARS-CoV-2 in a subject, comprising administering to asubject in need thereof a therapeutically effective amount of theantibody or antigen-binding fragment thereof or a therapeuticallyeffective amount of the pharmaceutical composition, as described above.

In another aspect, this disclosure additionally provides a method ofpreventing or treating a SARS-CoV-2 infection, comprising administeringto a subject in need thereof a therapeutically effective amount of theantibody or antigen-binding fragment thereof or a therapeuticallyeffective amount of the pharmaceutical composition, as described above.

The neutralizing of the SARS-CoV-2 virus can be done via (i) inhibitingSARS-CoV-2 virus binding to a target cell; (ii) inhibiting SARS-CoV-2virus uptake by a target cell; (iii) inhibiting SARS-CoV-2 virusreplication; and (iv) inhibiting SARS-CoV-2 virus particles release frominfected cells. One skilled in the art possesses the ability to performany assay to assess neutralization of SARS-CoV-2 virus.

Notably, the neutralizing properties of antibodies may be assessed by avariety of tests, which all may assess the consequences of (i)inhibition of SARS-CoV-2 virus binding to a target cell; (ii) inhibitionof SARS-CoV-2 virus uptake by a target cell; (iii) inhibition ofSARS-CoV-2 virus replication; and (iv) inhibition of SARS-CoV-2 virusparticles release from infected cells. In other words, implementingdifferent tests may lead to the observation of the same consequence,i.e., the loss of infectivity of the SARS-CoV-2 virus. Thus, in oneembodiment, the present invention provides a method of neutralizingSARS-CoV-2 virus in a subject comprising administering to the subject atherapeutically effective amount of the antibody of the presentinvention described herein.

Another aspect of the present invention provides a method of treating aSARS-CoV-2-related disease. Such a method includes therapeutic(following SARS-CoV-2 infection) and prophylactic (prior to SARS-CoV-2exposure, infection, or pathology). For example, therapeutic andprophylactic methods of treating an individual for a SARS-CoV-2infection include treatment of an individual having or at risk of havinga SARS-CoV-2 infection or pathology, treating an individual with aSARS-CoV-2 infection, and methods of protecting an individual from aSARS-CoV-2 infection, to decrease or reduce the probability of aSARS-CoV-2 infection in an individual, to decrease or reducesusceptibility of an individual to a SARS-CoV-2 infection, or to inhibitor prevent a SARS-CoV-2 infection in an individual, and to decrease,reduce, inhibit or suppress transmission of a SARS-CoV-2 from aninfected individual to an uninfected individual. Such methods includeadministering an antibody of the present invention or a compositioncomprising the antibody disclosed herein to therapeutically orprophylactically treat (vaccinate or immunize) an individual having orat risk of having a SARS-CoV-2 infection or pathology. Accordingly,methods can treat the SARS-CoV-2 infection or pathology, or provide theindividual with protection from infection (e.g., prophylacticprotection).

In one embodiment, a method of treating a SARS-CoV-2-related diseasecomprises administering to an individual in need thereof an antibody ortherapeutic composition disclosed herein in an amount sufficient toreduce one or more physiological conditions or symptoms associated witha SARS-CoV-2 infection or pathology, thereby treating theSARS-CoV-2-related disease.

In one embodiment, an antibody or therapeutic composition disclosedherein is used to treat a SARS-CoV-2-related disease. Use of an antibodyor therapeutic composition disclosed herein treats a SARS-CoV-2-relateddisease by reducing one or more physiological conditions or symptomsassociated with a SARS-CoV-2 infection or pathology. In aspects of thisembodiment, administration of an antibody or therapeutic compositiondisclosed herein is in an amount sufficient to reduce one or morephysiological conditions or symptoms associated with a SARS-CoV-2infection or pathology, thereby treating the SARS-CoV-2-based disease.In other aspects of this embodiment, administration of an antibody ortherapeutic composition disclosed herein is in an amount sufficient toincrease, induce, enhance, augment, promote or stimulate SARS-CoV-2clearance or removal; or decrease, reduce, inhibit, suppress, prevent,control, or limit transmission of SARS-CoV-2 to another individual.

One or more physiological conditions or symptoms associated with aSARS-CoV-2 infection or pathology will respond to a method of treatmentdisclosed herein. The symptoms of SARS-CoV-2 infection or pathologyvary, depending on the phase of infection.

In some embodiments, the method of neutralizing SARS-CoV-2 in a subjectcomprises administering to a subject in need thereof a therapeuticallyeffective amount of a first antibody or antigen-binding fragment thereofand a second antibody or antigen-binding fragment thereof of theantibody or antigen-binding fragment, as described above, wherein thefirst antibody or antigen-binding fragment thereof and the secondantibody or antigen binding fragment thereof exhibit synergisticactivity or a therapeutically effective amount of the pharmaceuticalcomposition described above.

In some embodiments, the method of preventing or treating a SARS-CoV-2infection, comprising administering to a subject in need thereof atherapeutically effective amount of a first antibody or antigen-bindingfragment thereof and a second antibody or antigen-binding fragmentthereof of the antibody or antigen-binding fragment, as described above,wherein the first antibody or antigen-binding fragment thereof and thesecond antibody or antigen binding fragment thereof exhibit synergisticactivity or a therapeutically effective amount of the pharmaceuticalcomposition described above. In some embodiments, the first antibody orantigen-binding fragment thereof is administered before, after, orconcurrently with the second antibody or antigen-binding fragmentthereof.

In some embodiments, the first antibody or antigen-binding fragmentthereof and the second antibody or antigen-binding fragment thereof canbe any combinations of the antibody or antigen-binding fragment thereofcomprising a heavy chain and a light chain that comprise the respectiveamino acid sequences described herein.

In some embodiments, the second therapeutic agent comprises ananti-inflammatory drug or an antiviral compound. In some embodiments,the antiviral compound comprises: a nucleoside analog, a peptoid, anoligopeptide, a polypeptide, a protease inhibitor, a 3C-like proteaseinhibitor, a papain-like protease inhibitor, or an inhibitor of an RNAdependent RNA polymerase. In some embodiments, the antiviral compoundmay include: acyclovir, gancyclovir, vidarabine, foscarnet, cidofovir,amantadine, ribavirin, trifluorothymidine, zidovudine, didanosine,zalcitabine, or an interferon. In some embodiments, the interferon is aninterferon-α or an interferon-(3.

In some embodiments, the antibody or antigen-binding fragment thereof isadministered before, after, or concurrently with the second therapeuticagent or therapy. In some embodiments, the antibody or antigen-bindingfragment thereof is administered to the subject intravenously,subcutaneously, or intraperitoneally. In some embodiments, the antibodyor antigen-binding fragment thereof is administered prophylactically ortherapeutically.

The antibodies described herein can be used together with one or more ofother anti-SARS-CoV-2 virus antibodies to neutralize SARS-CoV-2 virusand thereby treating SARS-CoV-2 infections.

Combination Therapies

Combination therapies may include an anti-SARS-CoV-2 antibody of theinvention and any additional therapeutic agent that may beadvantageously combined with an antibody of the invention or with abiologically active fragment of an antibody of the invention. Theantibodies of the present invention may be combined synergistically withone or more drugs or therapy used to treat a disease or disorderassociated with a viral infection, such as a SARS-CoV-2 infection. Insome embodiments, the antibodies of the invention may be combined with asecond therapeutic agent to ameliorate one or more symptoms of saiddisease. In some embodiments, the antibodies of the invention may becombined with a second antibody to provide synergistic activity inameliorating one or more symptoms of said disease. In some embodiments,the first antibody or antigen-binding fragment thereof is administeredbefore, after, or concurrently with the second antibody orantigen-binding fragment thereof.

For example, the antibody described herein can be used in variousdetection methods for use in, e.g., monitoring the progression of aSARS-CoV-2 infection; monitoring patient response to treatment for suchan infection, etc. The present disclosure provides methods of detectinga neuraminidase polypeptide in a biological sample obtained from anindividual. The methods generally involve: a) contacting the biologicalsample with a subject anti-neuraminidase antibody; and b) detectingbinding, if any, of the antibody to an epitope present in the sample. Insome instances, the antibody comprises a detectable label. The level ofneuraminidase polypeptide detected in the biological sample can providean indication of the stage, degree, or severity of a SARS-CoV-2infection. The level of the neuraminidase polypeptide detected in thebiological sample can provide an indication of the individual's responseto treatment for a SARS-CoV-2 infection.

In some embodiments, the second therapeutic agent is another antibody toa SARS-COV-2 protein or a fragment thereof. It is contemplated herein touse a combination (“cocktail”) of antibodies with broad neutralizationor inhibitory activity against SARS-COV-2. In some embodiments,non-competing antibodies may be combined and administered to a subjectin need thereof. In some embodiments, the antibodies comprising thecombination bind to distinct non-overlapping epitopes on the protein. Insome embodiments, the second antibody may possess longer half-life inhuman serum.

As used herein, the term “in combination with” means that additionaltherapeutically active component(s) may be administered prior to,concurrent with, or after the administration of the anti-SARS-COV-2antibody of the present invention. The term “in combination with” alsoincludes sequential or concomitant administration of an anti-SARS-COV-2antibody and a second therapeutic agent.

The additional therapeutically active component(s) may be administeredto a subject prior to administration of an anti-SARS-COV-2 antibody ofthe present invention. For example, a first component may be deemed tobe administered “prior to” a second component if the first component isadministered 1 week before, 72 hours before, 60 hours before, 48 hoursbefore, 36 hours before, 24 hours before, 12 hours before, 6 hoursbefore, 5 hours before, 4 hours before, 3 hours before, 2 hours before,1 hour before, 30 minutes before, 15 minutes before, 10 minutes before,5 minutes before, or less than 1 minute before administration of thesecond component. In other embodiments, the additional therapeuticallyactive component(s) may be administered to a subject afteradministration of an anti-SARS-COV-2 antibody of the present invention.For example, a first component may be deemed to be administered “after”a second component if the first component is administered 1 minuteafter, 5 minutes after, 10 minutes after, 15 minutes after, 30 minutesafter, 1 hour after, 2 hours after, 3 hours after, 4 hours after, 5hours after, 6 hours after, 12 hours after, 24 hours after, 36 hoursafter, 48 hours after, 60 hours after, 72 hours after administration ofthe second component. In yet other embodiments, the additionaltherapeutically active component(s) may be administered to a subjectconcurrent with administration of an anti-SARS-COV-2 antibody of thepresent invention. “Concurrent” administration, for purposes of thepresent invention, includes, e.g., administration of an anti-SARS-COV-2antibody and an additional therapeutically active component to a subjectin a single dosage form, or in separate dosage forms administered to thesubject within about 30 minutes or less of each other. If administeredin separate dosage forms, each dosage form may be administered via thesame route (e.g., both the anti-SARS-COV-2 antibody and the additionaltherapeutically active component may be administered intravenously,etc.); alternatively, each dosage form may be administered via adifferent route (e.g., the anti-SARS-COV-2 antibody may be administeredintravenously, and the additional therapeutically active component maybe administered orally). In any event, administering the components in asingle dosage from, in separate dosage forms by the same route, or inseparate dosage forms by different routes are all considered “concurrentadministration,” for purposes of the present disclosure. For purposes ofthe present disclosure, administration of an anti-SARS-COV-2 antibody“prior to,” “concurrent with,” or “after” (as those terms are definedhereinabove) administration of an additional therapeutically activecomponent is considered administration of an anti-SARS-COV-2 antibody“in combination with” an additional therapeutically active component.

The present invention includes pharmaceutical compositions in which ananti-SARS-COV-2 antibody of the present invention is co-formulated withone or more of the additional therapeutically active component(s) asdescribed elsewhere herein.

Administration Regimens

According to certain embodiments, a single dose of an anti-SARS-COV-2antibody of the invention (or a pharmaceutical composition comprising acombination of an anti-SARS-COV-2 antibody and any of the additionaltherapeutically active agents mentioned herein) may be administered to asubject in need thereof. According to certain embodiments of the presentinvention, multiple doses of an anti-SARS-COV-2 antibody (or apharmaceutical composition comprising a combination of ananti-SARS-COV-2 antibody and any of the additional therapeuticallyactive agents mentioned herein) may be administered to a subject over adefined time course. The methods according to this aspect of theinvention comprise sequentially administering to a subject multipledoses of an anti-SARS-COV-2 antibody of the invention. As used herein,“sequentially administering” means that each dose of anti-SARS-COV-2antibody is administered to the subject at a different point in time,e.g., on different days separated by a predetermined interval (e.g.,hours, days, weeks or months). The present invention includes methodswhich comprise sequentially administering to the patient a singleinitial dose of an anti-SARS-COV-2 antibody, followed by one or moresecondary doses of the anti-SARS-COV-2 antibody, and optionally followedby one or more tertiary doses of the anti-SARS-COV-2 antibody.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the anti-SARS-COV-2antibody of the invention. Thus, the “initial dose” is the dose which isadministered at the beginning of the treatment regimen (also referred toas the “baseline dose”); the “secondary doses” are the doses which areadministered after the initial dose; and the “tertiary doses” are thedoses which are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount ofanti-SARS-COV-2 antibody, but generally may differ from one another interms of frequency of administration. In some embodiments, however, theamount of anti-SARS-COV-2 antibody contained in the initial, secondaryand/or tertiary doses varies from one another (e.g., adjusted up or downas appropriate) during the course of treatment. In some embodiments, twoor more (e.g., 2, 3, 4, or 5) doses are administered at the beginning ofthe treatment regimen as “loading doses” followed by subsequent dosesthat are administered on a less frequent basis (e.g., “maintenancedoses”).

In certain exemplary embodiments of the present invention, eachsecondary and/or tertiary dose is administered 1 to 48 hours (e.g., 1,1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11,11½, 12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19,19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, ormore) after the immediately preceding dose. The phrase “the immediatelypreceding dose,” as used herein, means, in a sequence of multipleadministrations, the dose of anti-SARS-COV-2 antibody, which isadministered to a patient prior to the administration of the very nextdose in the sequence with no intervening doses.

The methods, according to this aspect of the invention, may compriseadministering to a patient any number of secondary and/or tertiary dosesof an anti-SARS-COV-2 antibody. For example, In some embodiments, only asingle secondary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondarydoses are administered to the patient. Likewise, In some embodiments,only a single tertiary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiarydoses are administered to the patient.

In some embodiments of the invention, the frequency at which thesecondary and/or tertiary doses are administered to a patient can varyover the course of the treatment regimen. The frequency ofadministration may also be adjusted during the course of treatment by aphysician depending on the needs of the individual patient followingclinical examination.

Diagnostic Uses of the Antibodies

The anti-SARS-COV-2 antibodies of the present invention may be used todetect and/or measure SARS-COV-2 in a sample, e.g., for diagnosticpurposes. Some embodiments contemplate the use of one or more antibodiesof the present invention in assays to detect aSARS-COV-2-associated-disease or disorder. Exemplary diagnostic assaysfor SARS-COV-2 may comprise, e.g., contacting a sample, obtained from apatient, with an anti-SARS-COV-2 antibody of the invention, wherein theanti-SARS-COV-2 antibody is labeled with a detectable label or reportermolecule or used as a capture ligand to selectively isolate SARS-COV-2from patient samples. Alternatively, an unlabeled anti-SARS-COV-2antibody can be used in diagnostic applications in combination with asecondary antibody, which is itself detectably labeled. The detectablelabel or reporter molecule can be a radioisotope, such as H, C, P, S, orI; a fluorescent or chemiluminescent moiety such as fluoresceinisothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase,β-galactosidase, horseradish peroxidase, or luciferase. Specificexemplary assays that can be used to detect or measure SARS-COV-2 in asample include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

In another aspect, this disclosure further provides a method fordetecting the presence of SARS CoV-2 in a sample comprising the stepsof: (i) contacting a sample with the antibody or antigen-bindingfragment thereof described above; and (ii) determining binding of theantibody or antigen-binding fragment to one or more SARS CoV-2 antigens,wherein binding of the antibody to the one or more SARS CoV-2 antigensis indicative of the presence of SARS CoV-2 in the sample.

In some embodiments, the SARS-CoV-2 antigen comprises a S polypeptide,such as a S polypeptide of a human or an animal SARS-CoV-2. In someembodiments, the SARS-CoV-2 antigen comprises the receptor-bindingdomain (RBD) of the S polypeptide. In some embodiments, the RBDcomprises amino acids 319-541 of the S polypeptide.

In some embodiments, the antibody or antigen-binding fragment thereof isconjugated to a label. In some embodiments, the step of detectingcomprises contacting a secondary antibody with the antibody orantigen-binding fragment thereof and wherein the secondary antibodycomprises a label. In some embodiments, the label includes a fluorescentlabel, a chemiluminescent label, a radiolabel, and an enzyme.

In some embodiments, the step of detecting comprises detectingfluorescence or chemiluminescence. In some embodiments, the step ofdetecting comprises a competitive binding assay or ELISA.

In some embodiments, the method further comprises binding the sample toa solid support. In some embodiments, the solid support includesmicroparticles, microbeads, magnetic beads, and an affinity purificationcolumn.

Samples that can be used in SARS-COV-2 diagnostic assays according tothe present invention include any tissue or fluid sample obtainable froma patient, which contains detectable quantities of either SARS-COV-2protein, or fragments thereof, under normal or pathological conditions.Generally, levels of SARS-COV-2 protein in a particular sample obtainedfrom a healthy patient (e.g., a patient not afflicted with a diseaseassociated with SARS-COV-2) will be measured to initially establish abaseline, or standard, level of SARS-COV-2. This baseline level ofSARS-COV-2 can then be compared against the levels of SARS-COV-2measured in samples obtained from individuals suspected of having aSARS-COV-2-associated condition, or symptoms associated with suchcondition.

The antibodies specific for SARS-COV-2 protein may contain no additionallabels or moieties, or they may contain an N-terminal or C-terminallabel or moiety. In one embodiment, the label or moiety is biotin. In abinding assay, the location of a label (if any) may determine theorientation of the peptide relative to the surface upon which thepeptide is bound. For example, if a surface is coated with avidin, apeptide containing an N-terminal biotin will be oriented such that theC-terminal portion of the peptide will be distal to the surface.

Definitions

To aid in understanding the detailed description of the compositions andmethods according to the disclosure, a few express definitions areprovided to facilitate an unambiguous disclosure of the various aspectsof the disclosure. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this disclosurebelongs.

The term “antibody” as referred to herein includes whole antibodies andany antigen-binding fragment or single chains thereof. Whole antibodiesare glycoproteins comprising at least two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. Each heavy chain iscomprised of a heavy chain variable region (abbreviated herein as VH)and a heavy chain constant region. The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as VL)and a light chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The heavy chain variable region CDRs and FRs are HFR1, HCDR1,HFR2, HCDR2, HFR3, HCDR3, HFR4. The light chain variable region CDRs andFRs are LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, LFR4. The variableregions of the heavy and light chains contain a binding domain thatinteracts with an antigen. The constant regions of the antibodies canmediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (e.g., effector cells) andthe first component (CIq) of the classical complement system.

The term “antigen-binding fragment or portion” of an antibody (or simply“antibody fragment or portion”), as used herein, refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., a Spike or S protein of SARS-CoV-2 virus). It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding fragment or portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CHI domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fab′ fragment, which is essentially a Fab withpart of the hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3rd ed.1993)); (iv) a Fd fragment consisting of the VH and CHI domains; (v) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (vi) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a VH domain; (vii) an isolated CDR; and (viii) ananobody, a heavy chain variable region containing a single variabledomain and two constant domains. Furthermore, although the two domainsof the Fv fragment, VL and VH, are coded for by separate genes, they canbe joined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv or scFv);see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al.(1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chainantibodies are also intended to be encompassed within the term“antigen-binding fragment or portion” of an antibody. These antibodyfragments are obtained using conventional techniques known to those withskill in the art, and the fragments are screened for utility in the samemanner as are intact antibodies.

An “isolated antibody,” as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds to a Spike or S protein of SARS-CoV-2 virus is substantially freeof antibodies that specifically bind antigens other than theneuraminidase). An isolated antibody can be substantially free of othercellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody” is intended to include antibodies havingvariable regions in which both the framework and CDR regions are derivedfrom human germline immunoglobulin sequences. Furthermore, if theantibody contains a constant region, the constant region also is derivedfrom human germline immunoglobulin sequences. The human antibodies ofthe invention can include amino acid residues not encoded by humangermline immunoglobulin sequences (e.g., mutations introduced by randomor site-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody,” as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity, which have variable regions in which boththe framework and CDR regions are derived from human germlineimmunoglobulin sequences. In one embodiment, the human monoclonalantibodies can be produced by a hybridoma that includes a B cellobtained from a transgenic nonhuman animal, e.g., a transgenic mouse,having a genome comprising a human heavy chain transgene and a lightchain transgene fused to an immortalized cell.

The term “recombinant human antibody,” as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further below), (b)antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In some embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

The term “isotype” refers to the antibody class (e.g., IgM or IgG1) thatis encoded by the heavy chain constant region genes. The phrases “anantibody recognizing an antigen” and “an antibody specific for anantigen” are used interchangeably herein with the term “an antibodywhich binds specifically to an antigen.”

The term “human antibody derivatives” refers to any modified form of thehuman antibody, e.g., a conjugate of the antibody and another agent orantibody. The term “humanized antibody” is intended to refer toantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto humanframework sequences. Additional framework region modifications can bemade within the human framework sequences.

The term “chimeric antibody” is intended to refer to antibodies in whichthe variable region sequences are derived from one species, and theconstant region sequences are derived from another species, such as anantibody in which the variable region sequences are derived from a mouseantibody, and the constant region sequences are derived from a humanantibody. The term can also refer to an antibody in which its variableregion sequence or CDR(s) is derived from one source (e.g., an IgA1antibody) and the constant region sequence or Fc is derived from adifferent source (e.g., a different antibody, such as an IgG, IgA2, IgD,IgE or IgM antibody).

The invention encompasses isolated or substantially purified nucleicacids, peptides, polypeptides or proteins. In the context of the presentinvention, an “isolated” nucleic acid, DNA or RNA molecule or an“isolated” polypeptide is a nucleic acid, DNA molecule, RNA molecule, orpolypeptide that exists apart from its native environment and istherefore not a product of nature. An isolated nucleic acid, DNAmolecule, RNA molecule or polypeptide may exist in a purified form ormay exist in a non-native environment such as, for example, a transgenichost cell. A “purified” nucleic acid molecule, peptide, polypeptide orprotein, or a fragment thereof, is substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. In one embodiment, an “isolated” nucleic acid isfree of sequences that naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For example, invarious embodiments, the isolated nucleic acid molecule can contain lessthan about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotidesequences that naturally flank the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived. A protein, peptideor polypeptide that is substantially free of cellular material includespreparations of protein, peptide or polypeptide having less than about30%, 20%, 10%, or 5% (by dry weight) of contaminating protein. When theprotein of the invention, or biologically active portion thereof, isrecombinantly produced, preferably culture medium represents less thanabout 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors ornon-protein-of-interest chemicals.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, pegylation, or any other manipulation,such as conjugation with a labeling component. As used herein, the term“amino acid” includes natural and/or unnatural or synthetic amino acids,including glycine and both the D or L optical isomers, and amino acidanalogs and peptidomimetics.

A peptide or polypeptide “fragment” as used herein refers to a less thanfull-length peptide, polypeptide or protein. For example, a peptide orpolypeptide fragment can have is at least about 3, at least about 4, atleast about 5, at least about 10, at least about 20, at least about 30,at least about 40 amino acids in length, or single unit lengths thereof.For example, fragment may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,or more amino acids in length. There is no upper limit to the size of apeptide fragment. However, in some embodiments, peptide fragments can beless than about 500 amino acids, less than about 400 amino acids, lessthan about 300 amino acids or less than about 250 amino acids in length.Preferably the peptide fragment can elicit an immune response when usedto inoculate an animal. A peptide fragment may be used to elicit animmune response by inoculating an animal with a peptide fragment incombination with an adjuvant, a peptide fragment that is coupled to anadjuvant, or a peptide fragment that is coupled to arsanilic acid,sulfanilic acid, an acetyl group, or a picryl group. A peptide fragmentcan include a non-amide bond and can be a peptidomimetic.

As used herein, the term “conjugate” or “conjugation” or “linked” asused herein refers to the attachment of two or more entities to form oneentity. A conjugate encompasses both peptide-small molecule conjugatesas well as peptide-protein/peptide conjugates.

The term “recombinant,” as used herein, refers to antibodies orantigen-binding fragments thereof of the invention created, expressed,isolated or obtained by technologies or methods known in the art asrecombinant DNA technology which include, e.g., DNA splicing andtransgenic expression. The term refers to antibodies expressed in anon-human mammal (including transgenic non-human mammals, e.g.,transgenic mice), or a cell (e.g., CHO cells) expression system orisolated from a recombinant combinatorial human antibody library.

A “nucleic acid” or “polynucleotide” refers to a DNA molecule (forexample, but not limited to, a cDNA or genomic DNA) or an RNA molecule(for example, but not limited to, an mRNA), and includes DNA or RNAanalogs. A DNA or RNA analog can be synthesized from nucleotide analogs.The DNA or RNA molecules may include portions that are not naturallyoccurring, such as modified bases, modified backbone,deoxyribonucleotides in an RNA, etc. The nucleic acid molecule can besingle-stranded or double-stranded.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 90%, and more preferablyat least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or GAP, as discussed below. A nucleic acid molecule havingsubstantial identity to a reference nucleic acid molecule may, incertain instances, encode a polypeptide having the same or substantiallysimilar amino acid sequence as the polypeptide encoded by the referencenucleic acid molecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 90% sequence identity, even more preferably atleast 95%, 98% or 99% sequence identity. Preferably, residue positions,which are not identical, differ by conservative amino acidsubstitutions. A “conservative amino acid substitution” is one in whichan amino acid residue is substituted by another amino acid residuehaving a side chain (R group) with similar chemical properties (e.g.,charge or hydrophobicity). In general, a conservative amino acidsubstitution will not substantially change the functional properties ofa protein. In cases where two or more amino acid sequences differ fromeach other by conservative substitutions, the percent or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment are wellknown to those of skill in the art. See, e.g., Pearson (1994) MethodsMol. Biol. 24: 307-331, which is herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include 1) aliphatic side chains: glycine, alanine,valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains:serine and threonine; 3) amide-containing side chains: asparagine andglutamine; 4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; 5) basic side chains: lysine, arginine, and histidine; 6)acidic side chains: aspartate and glutamate, and 7) sulfur-containingside chains: cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443 45, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides is typically measured usingsequence analysis software. Protein analysis software matches similarsequences using measures of similarity assigned to varioussubstitutions, deletions, and other modifications, includingconservative amino acid substitutions. For instance, GCG softwarecontains programs such as GAP and BESTFIT, which can be used withdefault parameters to determine sequence homology or sequence identitybetween closely related polypeptides, such as homologous polypeptidesfrom different species of organisms or between a wild type protein and amutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences alsocan be compared using FASTA with default or recommended parameters; aprogram in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences (Pearson (2000) supra).Another preferred algorithm when comparing a sequence of the inventionto a database containing a large number of sequences from differentorganisms is the computer program BLAST, especially BLASTP or TBLASTN,using default parameters. See, e.g., Altschul et al. (1990) J. Mol.Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each ofwhich is herein incorporated by reference.

As used herein, the term “affinity” refers to the strength of the sumtotal of noncovalent interactions between a single binding site of amolecule (e.g., an antibody) and its binding partner (e.g., an antigen).Unless indicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity, which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (KD). Affinity can be measured by common methodsknown in the art, including those described herein.

The term “specifically binds,” or “binds specifically to,” or the like,refers to an antibody that binds to a single epitope, e.g., underphysiologic conditions, but which does not bind to more than oneepitope. Accordingly, an antibody that specifically binds to apolypeptide will bind to an epitope that present on the polypeptide, butwhich is not present on other polypeptides. Specific binding can becharacterized by an equilibrium dissociation constant of at least about1×10-8 M or less (e.g., a smaller KD denotes a tighter binding). Methodsfor determining whether two molecules specifically bind are well knownin the art and include, for example, equilibrium dialysis, surfaceplasmon resonance, and the like. As described herein, antibodies havebeen identified by surface plasmon resonance, e.g., BIACORE™, which bindspecifically to a Spike or S protein of SARS-CoV-2 virus.

Preferably, the antibody binds to a Spike or S protein with “highaffinity,” namely with a KD of 1×10-7 M or less, more preferably 5×10-8M or less, more preferably 3×10-8 M or less, more preferably 1×10-8 M orless, more preferably 5×10-9 M or less or even more preferably 1×10-9 Mor less, as determined by surface plasmon resonance, e.g., BIACORE. Theterm “does not substantially bind” to a protein or cells, as usedherein, means does not bind or does not bind with a high affinity to theprotein or cells, i.e., binds to the protein or cells with a KD of1×10-6 M or more, more preferably 1×10-5 M or more, more preferably1×10-4 M or more, more preferably 1×10-3 M or more, even more preferably1×10-2 M or more.

The term “Kassoc” or “Ka,” as used herein, is intended to refer to theassociation rate of a particular antibody-antigen interaction, whereasthe term “Kdis” or “Kd,” as used herein, is intended to refer to thedissociation rate of a particular antibody-antigenn interaction. Theterm “KD,” as used herein, is intended to refer to the dissociationconstant, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) andis expressed as a molar concentration (M). KD values for antibodies canbe determined using methods well established in the art. A preferredmethod for determining the KD of an antibody is by using surface plasmonresonance, preferably using a biosensor system such as a BIACORE system.

Antibodies that “compete with another antibody for binding to a target”refer to antibodies that inhibit (partially or completely) the bindingof the other antibody to the target. Whether two antibodies compete witheach other for binding to a target, i.e., whether and to what extent oneantibody inhibits the binding of the other antibody to a target, may bedetermined using known competition experiments. In some embodiments, anantibody competes with, and inhibits binding of another antibody to atarget by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.The level of inhibition or competition may be different depending onwhich antibody is the “blocking antibody” (i.e., the cold antibody thatis incubated first with the target). Competition assays can be conductedas described, for example, in Ed Harlow and David Lane, Cold Spring HarbProtoc; 2006 or in Chapter 11 of “Using Antibodies” by Ed Harlow andDavid Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., USA 1999. Competing antibodies bind to the same epitope, anoverlapping epitope or to adjacent epitopes (e.g., as evidenced bysteric hindrance). Other competitive binding assays include: solid phasedirect or indirect radioimmunoassay (RIA), solid phase direct orindirect enzyme immunoassay (EIA), sandwich competition assay (seeStahli et al., Methods in Enzymology 9:242 (1983)); solid phase directbiotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986));solid phase direct labeled assay, solid phase direct labeled sandwichassay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Press (1988)); solid phase direct label RIA using 1-125 label(see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase directbiotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and directlabeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

The term “epitope” as used herein refers to an antigenic determinantthat interacts with a specific antigen-binding site in the variableregion of an antibody molecule known as a paratope. A single antigen mayhave more than one epitope. Thus, different antibodies may bind todifferent areas on an antigen and may have different biological effects.The term “epitope” also refers to a site on an antigen to which B and/orT cells respond. It also refers to a region of an antigen that is boundby an antibody. Epitopes may be defined as structural or functional.Functional epitopes are generally a subset of the structural epitopesand have those residues that directly contribute to the affinity of theinteraction. Epitopes may also be conformational, that is, composed ofnon-linear amino acids. In some embodiments, epitopes may includedeterminants that are chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl groups, or sulfonylgroups, and, In some embodiments, may have specific three-dimensionalstructural characteristics, and/or specific charge characteristics. Anepitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14 or 15 amino acids in a unique spatial conformation. Methods fordetermining what epitopes are bound by a given antibody (i.e., epitopemapping) are well known in the art and include, for example,immunoblotting and immune-precipitation assays, wherein overlapping orcontiguous peptides from a Spike or S protein are tested for reactivitywith a given antibody. Methods of determining spatial conformation ofepitopes include techniques in the art and those described herein, forexample, x-ray crystallography and 2-dimensional nuclear magneticresonance (see, e.g., Epitope Mapping Protocols in Methods in MolecularBiology, Vol. 66, G. E. Morris, Ed. (1996)).

The term “epitope mapping” refers to the process of identification ofthe molecular determinants for antibody-antigen recognition.

The term “binds to an epitope” or “recognizes an epitope” with referenceto an antibody or antibody fragment refers to continuous ordiscontinuous segments of amino acids within an antigen. Those of skillin the art understand that the terms do not necessarily mean that theantibody or antibody fragment is in direct contact with every amino acidwithin an epitope sequence.

The term “binds to the same epitope” with reference to two or moreantibodies means that the antibodies bind to the same, overlapping orencompassing continuous or discontinuous segments of amino acids. Thoseof skill in the art understand that the phrase “binds to the sameepitope” does not necessarily mean that the antibodies bind to orcontact exactly the same amino acids. The precise amino acids that theantibodies contact can differ. For example, a first antibody can bind toa segment of amino acids that is completely encompassed by the segmentof amino acids bound by a second antibody. In another example, a firstantibody binds one or more segments of amino acids that significantlyoverlap the one or more segments bound by the second antibody. For thepurposes herein, such antibodies are considered to “bind to the sameepitope.”

As used herein, the term “immune response” refers to a biologicalresponse within a vertebrate against foreign agents, which responseprotects the organism against these agents and diseases caused by them.An immune response is mediated by the action of a cell of the immunesystem (for example, a T lymphocyte, B lymphocyte, natural killer (NK)cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil)and soluble macromolecules produced by any of these cells or the liver(including antibodies, cytokines, and complement) that results inselective targeting, binding to, damage to, destruction of, and/orelimination from the vertebrate's body of invading pathogens, cells ortissues infected with pathogens, cancerous or other abnormal cells, or,in cases of autoimmunity or pathological inflammation, normal humancells or tissues. An immune reaction includes, e.g., activation orinhibition of a T cell, e.g., an effector T cell or a Th cell, such as aCD4+ or CD8+ T cell, or the inhibition of a Treg cell.

The term “detectable label” as used herein refers to a molecule capableof detection, including, but not limited to, radioactive isotopes,fluorescers, chemiluminescers, chromophores, enzymes, enzyme substrates,enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions,metal sols, ligands (e.g., biotin, avidin, streptavidin or haptens),intercalating dyes and the like. The term “fluorescer” refers to asubstance or a portion thereof that is capable of exhibitingfluorescence in the detectable range.

In many embodiments, the terms “subject” and “patient” are usedinterchangeably irrespective of whether the subject has or is currentlyundergoing any form of treatment. As used herein, the terms “subject”and “subjects” may refer to any vertebrate, including, but not limitedto, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep,hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (forexample, a monkey, such as a cynomolgus monkey, chimpanzee, etc.) and ahuman). The subject may be a human or a non-human. In more exemplaryaspects, the mammal is a human. As used herein, the expression “asubject in need thereof” or “a patient in need thereof” means a human ornon-human mammal that exhibits one or more symptoms or indications ofdisorders (e.g., neuronal disorders, autoimmune diseases, andcardiovascular diseases), and/or who has been diagnosed withinflammatory disorders. In some embodiments, the subject is a mammal. Insome embodiments, the subject is human.

As used herein, the term “disease” is intended to be generallysynonymous and is used interchangeably with, the terms “disorder” and“condition” (as in medical condition), in that all reflect an abnormalcondition (e.g., inflammatory disorder) of the human or animal body orof one of its parts that impairs normal functioning, is typicallymanifested by distinguishing signs and symptoms, and causes the human oranimal to have a reduced duration or quality of life.

As used herein, the term “treating” or “treatment” of any disease ordisorder refers in one embodiment, to ameliorating the disease ordisorder (i.e., arresting or reducing the development of the disease orat least one of the clinical symptoms thereof). In another embodiment,“treating” or “treatment” refers to ameliorating at least one physicalparameter, which may not be discernible by the patient. In yet anotherembodiment, “treating” or “treatment” refers to modulating the diseaseor disorder, either physically, (e.g., stabilization of a discerniblesymptom), physiologically, (e.g., stabilization of a physicalparameter), or both. In yet another embodiment, “treating” or“treatment” refers to preventing or delaying the onset or development orprogression of the disease or disorder.

The terms “prevent,” “preventing,” “prevention,” “prophylactictreatment” and the like refer to reducing the probability of developinga disorder or condition in a subject, who does not have, but is at riskof or susceptible to developing a disorder or condition.

The terms “decrease,” “reduced,” “reduction,” “decrease,” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, “reduced,”“reduction” or “decrease” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example, a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% decrease(e.g., absent level as compared to a reference sample), or any decreasebetween 10-100% as compared to a reference level.

As used herein, the term “agent” denotes a chemical compound, a mixtureof chemical compounds, a biological macromolecule (such as a nucleicacid, an antibody, a protein or portion thereof, e.g., a peptide), or anextract made from biological materials such as bacteria, plants, fungi,or animal (particularly mammalian) cells or tissues. The activity ofsuch agents may render it suitable as a “therapeutic agent,” which is abiologically, physiologically, or pharmacologically active substance (orsubstances) that acts locally or systemically in a subject.

As used herein, the terms “therapeutic agent,” “therapeutic capableagent,” or “treatment agent” are used interchangeably and refer to amolecule or compound that confers some beneficial effect uponadministration to a subject. The beneficial effect includes enablementof diagnostic determinations; amelioration of a disease, symptom,disorder, or pathological condition; reducing or preventing the onset ofa disease, symptom, disorder, or condition; and generally counteractinga disease, symptom, disorder or pathological condition.

The term “therapeutic effect” is art-recognized and refers to a local orsystemic effect in animals, particularly mammals, and more particularlyhumans caused by a pharmacologically active substance.

The term “effective amount,” “effective dose,” or “effective dosage” isdefined as an amount sufficient to achieve or at least partially achievea desired effect. A “therapeutically effective amount” or“therapeutically effective dosage” of a drug or therapeutic agent is anyamount of the drug that, when used alone or in combination with anothertherapeutic agent, promotes disease regression evidenced by a decreasein severity of disease symptoms, an increase in frequency and durationof disease symptom-free periods, or a prevention of impairment ordisability due to the disease affliction. A “prophylactically effectiveamount” or a “prophylactically effective dosage” of a drug is an amountof the drug that, when administered alone or in combination with anothertherapeutic agent to a subject at risk of developing a disease or ofsuffering a recurrence of disease, inhibits the development orrecurrence of the disease. The ability of a therapeutic or prophylacticagent to promote disease regression or inhibit the development orrecurrence of the disease can be evaluated using a variety of methodsknown to the skilled practitioner, such as in human subjects duringclinical trials, in animal model systems predictive of efficacy inhumans, or by assaying the activity of the agent in in vitro assays.

Doses are often expressed in relation to bodyweight. Thus, a dose whichis expressed as [g, mg, or other unit]/kg (or g, mg etc.) usually refersto [g, mg, or other unit] “per kg (or g, mg etc.) bodyweight,” even ifthe term “bodyweight” is not explicitly mentioned.

As used herein, the term “composition” or “pharmaceutical composition”refers to a mixture of at least one component useful within theinvention with other components, such as carriers, stabilizers,diluents, dispersing agents, suspending agents, thickening agents,and/or excipients. The pharmaceutical composition facilitatesadministration of one or more components of the invention to anorganism.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the composition, and is relativelynon-toxic, i.e., the material may be administered to an individualwithout causing undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, the term “pharmaceutically acceptable carrier” includesa pharmaceutically acceptable salt, pharmaceutically acceptablematerial, composition or carrier, such as a liquid or solid filler,diluent, excipient, solvent or encapsulating material, involved incarrying or transporting a compound(s) of the present invention withinor to the subject such that it may perform its intended function.Typically, such compounds are carried or transported from one organ, orportion of the body, to another organ, or portion of the body. Each saltor carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation and not injurious to thesubject. Some examples of materials that may serve as pharmaceuticallyacceptable carriers include: sugars, such as lactose, glucose, andsucrose; starches, such as corn starch and potato starch; cellulose, andits derivatives, such as sodium carboxymethyl cellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil, and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; diluent; granulating agent; lubricant; binder;disintegrating agent; wetting agent; emulsifier; coloring agent; releaseagent; coating agent; sweetening agent; flavoring agent; perfumingagent; preservative; antioxidant; plasticizer; gelling agent; thickener;hardener; setting agent; suspending agent; surfactant; humectant;carrier; stabilizer; and other non-toxic compatible substances employedin pharmaceutical formulations, or any combination thereof. As usedherein, “pharmaceutically acceptable carrier” also includes any and allcoatings, antibacterial and antifungal agents, and absorption delayingagents, and the like that are compatible with the activity of one ormore components of the invention, and are physiologically acceptable tothe subject. Supplementary active compounds may also be incorporatedinto the compositions.

“Combination” therapy, as used herein, unless otherwise clear from thecontext, is meant to encompass administration of two or more therapeuticagents in a coordinated fashion and includes, but is not limited to,concurrent dosing. Specifically, combination therapy encompasses bothco-administration (e.g., administration of a co-formulation orsimultaneous administration of separate therapeutic compositions) andserial or sequential administration, provided that administration of onetherapeutic agent is conditioned in some way on the administration ofanother therapeutic agent. For example, one therapeutic agent may beadministered only after a different therapeutic agent has beenadministered and allowed to act for a prescribed period of time. See,e.g., Kohrt et al. (2011) Blood 117:2423.

As used herein, the term “co-administration” or “co-administered” refersto the administration of at least two agent(s) or therapies to asubject. In some embodiments, the co-administration of two or moreagents/therapies is concurrent. In other embodiments, a firstagent/therapy is administered prior to a second agent/therapy. Those ofskill in the art understand that the formulations and/or routes ofadministration of the various agents/therapies used may vary.

As used herein, the term “contacting,” when used in reference to any setof components, includes any process whereby the components to becontacted are mixed into the same mixture (for example, are added intothe same compartment or solution), and does not necessarily requireactual physical contact between the recited components. The recitedcomponents can be contacted in any order or any combination (orsub-combination) and can include situations where one or some of therecited components are subsequently removed from the mixture, optionallyprior to addition of other recited components. For example, “contactingA with B and C” includes any and all of the following situations: (i) Ais mixed with C, then B is added to the mixture; (ii) A and B are mixedinto a mixture; B is removed from the mixture, and then C is added tothe mixture; and (iii) A is added to a mixture of B and C.

“Sample,” “test sample,” and “patient sample” may be usedinterchangeably herein. The sample can be a sample of serum, urineplasma, amniotic fluid, cerebrospinal fluid, cells, or tissue. Such asample can be used directly as obtained from a patient or can bepre-treated, such as by filtration, distillation, extraction,concentration, centrifugation, inactivation of interfering components,addition of reagents, and the like, to modify the character of thesample in some manner as discussed herein or otherwise as is known inthe art. The terms “sample” and “biological sample” as used hereingenerally refer to a biological material being tested for and/orsuspected of containing an analyte of interest such as antibodies. Thesample may be any tissue sample from the subject. The sample maycomprise protein from the subject.

As used herein, the term “in vitro” refers to events that occur in anartificial environment, e.g., in a test tube or reaction vessel, in cellculture, etc., rather than within a multi-cellular organism.

As used herein, the term “in vivo” refers to events that occur within amulti-cellular organism, such as a non-human animal.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise.

As used herein, the terms “including,” “comprising,” “containing,” or“having” and variations thereof are meant to encompass the items listedthereafter and equivalents thereof as well as additional subject matterunless otherwise noted.

As used herein, the phrases “in one embodiment,” “in variousembodiments,” “in some embodiments,” and the like are used repeatedly.Such phrases do not necessarily refer to the same embodiment, but theymay unless the context dictates otherwise.

As used herein, the terms “and/or” or “/” means any one of the items,any combination of the items, or all of the items with which this termis associated.

As used herein, the word “substantially” does not exclude “completely,”e.g., a composition which is “substantially free” from Y may becompletely free from Y. Where necessary, the word “substantially” may beomitted from the definition of the invention.

As used herein, the term “each,” when used in reference to a collectionof items, is intended to identify an individual item in the collectionbut does not necessarily refer to every item in the collection.Exceptions can occur if explicit disclosure or context clearly dictatesotherwise.

As used herein, the term “approximately” or “about,” as applied to oneor more values of interest, refers to a value that is similar to astated reference value. In some embodiments, the term “approximately” or“about” refers to a range of values that fall within 25%, 20%, 19%, 18%,17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, or less in either direction (greater than or less than) of thestated reference value unless otherwise stated or otherwise evident fromthe context (except where such number would exceed 100% of a possiblevalue). Unless indicated otherwise herein, the term “about” is intendedto include values, e.g., weight percents, proximate to the recited rangethat are equivalent in terms of the functionality of the individualingredient, the composition, or the embodiment.

As disclosed herein, a number of ranges of values are provided. It isunderstood that each intervening value, to the tenth of the unit of thelower limit, unless the context clearly dictates otherwise, between theupper and lower limits of that range is also specifically disclosed.Each smaller range between any stated value or intervening value in astated range and any other stated or intervening value in that statedrange is encompassed within the invention. The upper and lower limits ofthese smaller ranges may independently be included or excluded in therange, and each range where either, neither, or both limits are includedin the smaller ranges is also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

All methods described herein are performed in any suitable order unlessotherwise indicated herein or otherwise clearly contradicted by context.In regard to any of the methods provided, the steps of the method mayoccur simultaneously or sequentially. When the steps of the method occursequentially, the steps may occur in any order, unless noted otherwise.In cases in which a method comprises a combination of steps, each andevery combination or sub-combination of the steps is encompassed withinthe scope of the disclosure, unless otherwise noted herein.

Each publication, patent application, patent, and other reference citedherein is incorporated by reference in its entirety to the extent thatit is not inconsistent with the present disclosure. Publicationsdisclosed herein are provided solely for their disclosure prior to thefiling date of the present invention. Nothing herein is to be construedas an admission that the present invention is not entitled to antedatesuch publication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dates,which may need to be independently confirmed.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

EXAMPLES Example 1

This example describes the materials and methods used in the subsequentEXAMPLES below.

Study Participants.

To isolate and characterize anti-SARS-CoV-2 RBD antibodies fromvaccinees, a cohort of 20 individuals that participated in either theModerna or Pfizer/BioNTech phase 3 vaccine clinical trial and no priorhistory of SARS-CoV-2 infection were recruited at the NIH Blood Centerand the Rockefeller University Hospital for blood donation. Eligibleparticipants included adults at least 18 years of age with no knownheart, lung, kidney disease, or bleeding disorders, no history of HIV-1or malaria infection. All participants were asymptomatic at the time ofthe study visit and had received a complete two-dose regimen of eithermRNA vaccine. Informed consent was obtained from all participants, andthe study was conducted in accordance with Good Clinical Practice. Thestudy visits and blood draws were reviewed and approved under theNational Institutes of Health's Federalwide Assurance (FWA00005897) inaccordance with Federal regulations 45 CFR 46 and 21 CFR 5 by the NIHIntramural Research Program IRB committee (IRB #99CC0168, Collection andDistribution of Blood Components from Healthy Donors for In vitroResearch Use) and by the Institutional Review Board of the RockefellerUniversity (IRB #DRO-1006, Peripheral Blood of Coronavirus Survivors toIdentify Virus-Neutralizing Antibodies). For detailed participantcharacteristics, see Table 1.

Blood Samples Processing and Storage.

Samples collected at NIH were drawn from participants at the study visitand processed within 24 hours. Briefly, whole blood samples weresubjected to Ficoll gradient centrifugation after 1:1 dilution in PBS.Plasma and PBMC samples were obtained through phase separation of plasmalayer and Buffy coat phase, respectively. PBMCs were further preparedthrough centrifugation, red blood cells lysis, and washing steps, andstored in CellBanker cell freezing media (Amsbio). All samples werealiquoted and stored at −80° C. and shipped on dry ice. Prior toexperiments, aliquots of plasma samples were heat-inactivated (56° C.for 1 hour) and then stored at 4° C. Peripheral Blood Mononuclear Cells(PBMCs) obtained from samples collected at Rockefeller University werepurified as previously reported (Gaebler, C. et al. bioRxiv, (2020);Robbiani, D. F. et al. Nature 584, 437-442 (2020)) by gradientcentrifugation and stored in liquid nitrogen in the presence of FCS andDMSO. Heparinized plasma samples were aliquoted and stored at −20 □ C orless. Prior to experiments, aliquots of plasma samples wereheat-inactivated (56 □ C for 1 hour) and then stored at 4° C.

ELISAs

ELISAs to evaluate antibodies binding to SARS-CoV-2 S (BioHub), RBD, andadditional mutated RBDs were performed by coating high-binding96-half-well plates (Corning 3690) with 50 μl per well of a 1 μg/mlprotein solution in PBS overnight at 4° C. Plates were washed 6 timeswith washing buffer (1×PBS with 0.05% Tween-20 (Sigma-Aldrich)) andincubated with 170 μl per well blocking buffer (1×PBS with 2% BSA and0.05% Tween-20 (Sigma)) for 1 h at room temperature. Immediately afterblocking, monoclonal antibodies or plasma samples were added in PBS andincubated for 1 h at room temperature. Plasma samples were assayed at a1:66.6 (RU samples) or a 1:33.3 (NIH samples) starting dilution and 7additional threefold serial dilutions. Monoclonal antibodies were testedat 10 μg/ml starting concentration and 10 additional fourfold serialdilutions. Plates were washed 6 times with washing buffer and thenincubated with anti-human IgG, IgM, or IgA secondary antibody conjugatedto horseradish peroxidase (HRP) (Jackson Immuno Research 109-036-088109-035-129 and Sigma A0295) in blocking buffer at a 1:5,000 dilution(IgM and IgG) or 1:3,000 dilution (IgA). Plates were developed byaddition of the HRP substrate, TMB (ThermoFisher) for 10 min (plasmasamples) or 4 minutes (monoclonal antibodies), then the developingreaction was stopped by adding 50 μl 1 M H₂SO₄ and absorbance wasmeasured at 450 nm with an ELISA microplate reader (FluoStar Omega, BMGLabtech) with Omega and Omega MARS software for analysis. For plasmasamples, a positive control (plasma from participant COV72 (Gaebler, C.et al. bioRxiv, (2020); Robbiani, D. F. et al. Nature 584, 437-442(2020)), diluted 66.6-fold and with seven additional threefold serialdilutions in PBS) was added to every assay plate for validation. Theaverage of its signal was used for normalization of all of the othervalues on the same plate with Excel software before calculating the areaunder the curve using Prism V8.4 (GraphPad). For monoclonal antibodies,the EC50 was determined using four-parameter nonlinear regression(GraphPad Prism V8.4).

Expression of RBD Proteins

Mammalian expression vectors encoding the RBDs of SARS-CoV-2 (GenBankMN985325.1; S protein residues 319-539) and eight additional mutant RBDproteins (E484K, Q493R, R346S, N493K, N440K, V367F, A475V, S477N, andV483A) with an N-terminal human IL-2 or Mu phosphatase signal peptidewere previously described (Barnes, C. O. et al. Cell 182, 828-842 e816(2020)).

Cells and Viruses

293T/ACE2.cl122 and HT1080iACE2.cl14 cells (Schmidt, F. et al. J Exp Med217 (2020)) were cultured in Dulbecco's Modified Eagle Medium (DMEM)supplemented with 10% fetal bovine serum (FBS) at 37° C. and 5% CO₂.Cells were periodically tested for contamination with mycoplasma orretroviruses.

rVSV/SARS-CoV-2/GFP chimeric virus stocks were generated by infecting293T/ACE2.cl22 cells. The supernatant was harvested 1 day post infection(dpi), cleared of cellular debris, and stored at −80° C. A plaquepurified variant designated rVSV/SARS-CoV-2/GFP_(2E1) that encodesD215G/R683G substitutions was used in these studies.

Selection and Analysis of Antibody Escape Mutations

For the selection of monoclonal antibody-resistant spike variants, anrVSV/SARS-CoV-2/GFP_(2E1) (Schmidt, F. et al. J Exp Med 217 (2020))population containing 10⁶ infectious units was incubated with monoclonalantibodies (10 μg/ml passage 10-40 μg/ml for 1 hr at 37° C. Then, thevirus-antibody mixtures were incubated with 5×10⁵ 293 T/ACE2cl.22 cellsin 6-well plates. At 1 day post infection, media was replaced with freshmedia containing the equivalent concentrations of antibodies. Thesupernatant was harvested 2 days after infection, and 150 μl of thecleared supernatant was used to infect cells for passage 2, while 150 μlwas subjected to RNA extraction and sequencing.

For identification of putative antibody resistance mutations, RNA wasextracted using NucleoSpin 96 Virus Core Kit (Macherey-Nagel). The RNAwas reversed transcribed using the SuperScript VILO cDNA Synthesis Kit(Thermo Fisher Scientific). KOD Xtreme Hot Start DNA Polymerase(Millipore Sigma) was used for amplification of cDNA using primersflanking the S-encoding sequence. The PCR products were purified andsequenced as previously described (Weisblum, Y. et al. Elife 9 (2020)).Briefly, tagmentation reactions were performed using 1 ul diluted cDNA,0.25 μl Nextera TDE1 Tagment DNA enzyme (catalog no. 15027865), and 1.25μl TD Tagment DNA buffer (catalog no. 15027866; Illumina). Next, the DNAwas ligated to unique i5/i7 barcoded primer combinations using theIllumina Nextera XT Index Kit v2 and KAPA HiFi HotStart ReadyMix (2X;KAPA Biosystems) and purified using AmPure Beads XP (Agencourt), afterwhich the samples were pooled into one library and subjected topaired-end sequencing using Illumina MiSeq Nano 300 V2 cycle kits(Illumina) at a concentration of 12 pM.

For analysis of the sequencing data, the raw paired-end reads werepre-processed to remove trim adapter sequences and to remove low-qualityreads (Phred quality score <20) using BBDuk. Reads were mapped to therVSV/SARS-CoV-2/GFP sequence using Geneious Prime (Version 2020.1.2).Mutations were annotated using Geneious Prime, with a P-value cutoff of10⁻⁶.

SARS-CoV-2 Pseudotyped Reporter Virus

A panel of plasmids expressing RBD-mutant SAR-CoV-2 spike proteins inthe context of pSARS-CoV-2-S_(Δ19) have been described previously(Weisblum, Y. et al. Elife 9 (2020)). Additional substitutions wereintroduced using either PCR primer-mediated mutagenesis or withsynthetic gene fragments (IDT) followed by Gibson assembly. The mutantsE484K and KEN (K417N+E484K+N501Y) were constructed in the context of apSARS-CoV-2-S_(Δ19) variant with a mutation in the furin cleavage site(R683G). The NT₅₀s and IC50 of these pseudotypes were compared to awildtype SARS-CoV-2 (NC_045512) spike sequence carrying R683G in thesubsequent analyses, as appropriate.

Generation of SARS-CoV-2 pseudotyped HIV-1 particles was performed aspreviously described (Robbiani, D. F. et al. Nature 584, 437-442(2020)). Briefly, 293T cells were transfected with pNL4-3ΔEnv-nanolucand pSARS-CoV-2-S_(Δ19) and pseudotyped virus stocks were harvested 48hours after transfection, filtered, and stored at −80° C.

SARS-CoV-2 Pseudotype Neutralization Assays

Plasma or monoclonal antibodies from vaccine recipients were four-foldor five-fold serially diluted and then incubated with SARS-CoV-2pseudotyped HIV-1 reporter virus for 1 h at 37° C. The antibody andpseudotype virus mixture was added to 293 T_(Ace2) cells (Robbiani, D.F. et al. Nature 584, 437-442 (2020)) (for comparisons of plasma fromCOVID-19-convalescents and vaccine recipients) or HT1080ACE2.cl14 cells(for analysis of spike mutants with vaccine recipient plasma ormonoclonal antibodies). After 48 h, cells were washed with PBS and lysedwith Luciferase Cell Culture Lysis 5× reagent (Promega), and NanolucLuciferase activity in lysates was measured using the Nano-GloLuciferase Assay System (Promega) with the Glomax Navigator (Promega).The relative luminescence units were normalized to those derived fromcells infected with SARS-CoV-2 pseudotyped virus in the absence ofplasma or monoclonal antibodies. The half-maximal neutralization titersfor plasma (NT₅₀) or half-maximal and 90% inhibitory concentrations formonoclonal antibodies (IC₅₀ and IC₉₀, respectively) were determinedusing four-parameter nonlinear regression (least-squares regressionmethod without weighting; constraints: top=1, bottom=0) (GraphPadPrism).

Biotinylation of Viral Protein for Use in Flow Cytometry

Purified and Avi-tagged SARS-CoV-2 RBD was biotinylated using theBiotin-Protein Ligase-BIRA kit according to manufacturer's instructions(Avidity) as described before (Robbiani, D. F. et al. Nature 584,437-442 (2020)). Ovalbumin (Sigma, A5503-1G) was biotinylated using theEZ-Link Sulfo-NHS-LC-Biotinylation kit according to the manufacturer'sinstructions (Thermo Scientific). Biotinylated ovalbumin was conjugatedto streptavidin-BV711 (BD biosciences, 563262) and RBD tostreptavidin-PE (BD Biosciences, 554061) and streptavidin-AF647(Biolegend, 405237).

Flow Cytometry and Single Cell Sorting

Single-cell sorting by flow cytometry was performed as describedpreviously (Robbiani, D. F. et al. Nature 584, 437-442 (2020)). Briefly,peripheral blood mononuclear cells were enriched for B cells by negativeselection using a pan-B-cell isolation kit according to themanufacturer's instructions (Miltenyi Biotec, 130-101-638). The enrichedB cells were incubated in FACS buffer (1×PBS, 2% FCS, 1 mM EDTA) withthe following anti-human antibodies (all at 1:200 dilution):anti-CD20-PECy7 (BD Biosciences, 335793), anti-CD3-APC-eFluro 780(Invitrogen, 47-0037-41), anti-CD8-APC-eFluor 780 (Invitrogen,47-0086-42), anti-CD16-APC-eFluor 780 (Invitrogen, 47-0168-41),anti-CD14-APC-eFluor 780 (Invitrogen, 47-0149-42), as well as Zombie NIR(BioLegend, 423105) and fluorophore-labelled RBD and ovalbumin (Ova) for30 min on ice. Single CD3−CD8+CD14−CD16−CD20+Ova−RBD−PE+RBD−AF647+Bcells were sorted into individual wells of 96-well plates containing 4μl of lysis buffer (0.5×PBS, 10 mM DTT, 3,000 units/ml RNasinRibonuclease Inhibitors (Promega, N2615) per well using a FACS Aria IIIand FACSDiva software (Becton Dickinson) for acquisition and FlowJo foranalysis. The sorted cells were frozen on dry ice, and then stored at−80° C. or immediately used for subsequent RNA reverse transcription.

Antibody Sequencing, Cloning, and Expression

Antibodies were identified and sequenced as described previously(Robbiani, D. F. et al. Nature 584, 437-442 (2020)). In brief, RNA fromsingle cells was reverse-transcribed (SuperScript III ReverseTranscriptase, Invitrogen, 18080-044), and the cDNA was stored at −20°C. or used for subsequent amplification of the variable IGH, IGL, andIGK genes by nested PCR and Sanger sequencing. Sequence analysis wasperformed using MacVector. Amplicons from the first PCR reaction wereused as templates for sequence- and ligation-independent cloning intoantibody expression vectors. Recombinant monoclonal antibodies and Fabswere produced and purified as previously described (Robbiani, D. F. etal. Nature 584, 437-442 (2020)).

Cryo-EM Sample Preparation

Expression and purification of SARS-CoV-2 6P stabilized S trimers wasconducted as previously described ((Hsieh, C. L. et al. Science 369,1501-1505 (2020); Cohen, A. A. et al. bioRxiv,doi:10.1101/2020.11.17.387092 (2020)). Purified Fab and S 6P trimer wereincubated at a 1.1:1 molar ratio per protomer on ice for 30 minutesprior to deposition on a freshly glow-discharged 300 mesh, 1.2/1.3Quantifoil copper grid. Immediately before 3 μl of the complex wasapplied to the grid, fluorinated octyl-malotiside was added to the Fab-Scomplex to a final detergent concentration of 0.02% w/v, resulting in afinal complex concentration of 3 mg/ml. Samples were vitrified in 100%liquid ethane using a Mark IV Vitrobot after blotting for 3 s withWhatman No. 1 filter paper at 22° C. and 100% humidity.

Cryo-EM Data Collection and Processing

Data collection and processing followed a similar workflow to what hasbeen previously described in detail (Barnes, C. O. et al. Nature 588,682-687 (2020)). Micrographs were collected on a Talos Arcticatransmission electron microscope (Thermo Fisher) operating at 200 kV forall Fab-S complexes. Data were collected using SerialEM automated datacollection software (Mastronarde, D. N. J Struct Biol 152, 36-51 (2005))and movies were recorded with a K3 camera (Gatan). For all datasets,cryo-EM movies were patch motion corrected for beam-induced motionincluding dose-weighting within cryoSPARC v2.15 (Punjani, A., et al. NatMethods 14, 290-296 (2017).) after binning super resolution movies. Thenon-dose-weighted images were used to estimate CTF parameters usingcryoSPARC implementation of the Patch CTF job. Particles were pickedusing Blob picker and extracted 4× binned and 2D classified. Classaverages corresponding to distinct views with secondary structurefeatures were chosen, and ab initio models were generated. 3D classesthat showed features of a Fab-S complex were re-extracted, unbinned(0.869 A/pixel), and homogenously refined with C1 symmetry. Overallresolutions were reported based on gold standard FSC calculations.

Cryo-EM Structure Modeling and Refinement

Coordinates for initial complexes were generated by docking individualchains from reference structures into cryo-EM density using UCSF Chimera(Goddard, T. D. et al. Protein Sci 27, 14-25 (2018)) (S trimer: PDB7KXL, Fab: PDB 6XCA or 7K8P after trimming CDR3 loops and converting toa polyalanine model). Models were then refined into cryo-EM maps byrigid body and real space refinement in Phenix (Terwilliger, T. C., etal. Nat Methods 15, 905-908 (2018)) If the resolution allowed, partialCDR3 loops were built manually in Coot (Emsley, P., et al. ActaCrystallogr D Biol Crystallogr 66, 486-501(2010)) and then refined usingreal-space refinement in Phenix.

Computational Analyses of Antibody Sequences

Antibody sequences were trimmed based on quality and annotated usingIgblastn v.1.14. with IMGT domain delineation system. Annotation wasperformed systematically using Change-O toolkit v.0.4.540 (Gupta, N. T.et al. Bioinformatics 31, 3356-3358 (2015)). Heavy and light chainsderived from the same cell were paired, and clonotypes were assignedbased on their V and J genes using in-house R and Perl scripts (Extendeddata FIG. 2). All scripts and the data used to process antibodysequences are publicly available on GitHub(https://github.com/stratust/igpipeline).

The frequency distributions of human V genes in anti-SARS-CoV-2antibodies from this study were compared to 131,284,220 IgH and IgLsequences generated by and downloaded from cAb-Rep (Guo, Y., et al.Front Immunol 10, 2365 (2019).), a database of human shared BCRclonotypes available at https://cab-rep.c2b2.columbia.edu/. Based on the97 distinct V genes that make up the 4186 analyzed sequences from Igrepertoire of the 14 participants present in this study, the IgH and IgLsequences were selected from the database that are partially coded bythe same V genes and counted them according to the constant region. Thefrequencies shown in (FIG. 2e and Extended Data FIG. 3a ) are relativeto the source, and isotype analyzed. The two-sided binomial test wasused to check whether the number of sequences belonging to a specificIgHV or IgLV gene in the repertoire is different according to thefrequency of the same IgV gene in the database. Adjusted p-values werecalculated using the false discovery rate (FDR) correction. Significantdifferences are denoted with stars.

Nucleotide somatic hypermutation and CDR3 length were determined usingin-house R and Perl scripts. For somatic hypermutations, IGHV and IGLVnucleotide sequences were aligned against their closest germlines usingIgblastn, and the number of differences were considered nucleotidemutations. The average mutations for V genes were calculated by dividingthe sum of all nucleotide mutations across all participants by thenumber of sequences used for the analysis. To calculate the GRAVY scoresof hydrophobicity (Kyte, J. & Doolittle, R. F. J Mol Biol 157, 105-132(1982)), Guy H. R. Hydrophobicity scale was used based on free energy oftransfer (kcal/mole) implemented by the R package Peptides (theComprehensive R Archive Network repository;https://journal.r-project.org/archive/2015/RJ-2015-001/RJ-2015-001.pdf). 1405 heavy chain CDR3 amino acid sequences from this study and22,654,256 IGH CDR3 sequences from the public database of memory B cellreceptor sequences were used. The two-tailed Wilcoxon nonparametric testwas used to test whether there is a difference in hydrophobicitydistribution.

Data Availability Statement:

The raw sequencing data and computer scripts associated with FIG. 2 havebeen deposited at Github (https://github.com/stratust/igpipeline). Thisstudy also uses data from “A Public Database of Memory and Naive B-CellReceptor Sequences” (DeWitt, W. S. et al. PLoS One 11, e0160853 (2016)),PDB (6VYB and 6NB6) and from “High frequency of shared clonotypes inhuman B cell receptor repertoires” (Soto, C. et al. Nature 566, 398-402(2019)). Cryo-EM maps associated with data reported in this manuscriptare deposited in the Electron Microscopy Data Bank (EMDB:https://www.ebi.ac.uk/pdbe/emdb/).

Example 2

To date, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)has infected nearly 100 million individuals resulting in almost twomillion deaths. Many vaccines are being deployed to prevent the disease,including two novel mRNA-based vaccines (Gaebler, C. & Nussenzweig, M.C. Nature 586, 501-502 (2020); Krammer, F. Nature 586, 516-527 (2020)).These vaccines elicit neutralizing antibodies and appear to be safe andeffective, but the precise nature of the elicited antibodies is notknown. Here, the antibody and memory B cell responses in a cohort of 20volunteers who received either the Moderna (mRNA-1273) orPfizer-BioNTech (BNT162b2) vaccines were reported. Consistent with priorreports, 8 weeks after the second vaccine injection, the volunteersshowed high levels of IgM, and IgG anti-SARS-CoV-2 spike protein (S),receptor binding domain (RBD) binding titers. Moreover, the plasmaneutralizing activity and the relative numbers of RBD-specific memory Bcells were equivalent to individuals who recovered from naturalinfection. However, activity against SARS-CoV-2 variants encoding E484Kor N501Y or the K417N:E484K:N501Y combination was reduced by a small butsignificant margin. Consistent with this finding, vaccine-elicitedmonoclonal antibodies (mAbs) potently neutralize SARS-CoV-2, targeting anumber of different RBD epitopes. However, neutralization by 9 of the 17most potent mAbs tested was reduced or abolished by either K417N, E484K,or N501Y mutations. Notably, the same mutations were selected whenrecombinant vesicular stomatitis virus (rVSV)/SARS-CoV-2 S was culturedin the presence of the vaccine-elicited mAbs. Thus vaccine- andvirus-encoded S adopts similar conformations to induce equivalentanti-RBD antibodies. Taken together, the results indicate that theantibodies in clinical use should be tested against newly arisingvariants, and that mRNA vaccines may need to be updated periodically.

Between Oct. 19, 2020 and Jan. 15, 2021, 20 volunteers who received twodoses of the Moderna (n=14) or Pfizer mRNA (n=6) vaccines were recruitedfor blood donation and analyzed. Ages of the analyzed volunteers rangedfrom 29-69 years (median 43); 12 (60%) were male and 8 (40%) female. 16participants identified as Caucasian, 2 as Hispanic, and 1 as AfricanAmerican or Asian, respectively. The time from the second vaccination tosample collection varied between 3-14 weeks with an average of 8 weeks.None of the volunteers had a history of prior SARS-CoV-2 infection, andnone reported serious adverse events after vaccination (Table 1).

Vaccine Plasma Binding and Neutralizing Activity Against SARS-CoV-2

Plasma IgM and IgG anti-SARS-CoV-2 S and RBD were measured byenzyme-linked immunosorbent assay (ELISA). All individuals tested showedreactivity to S and RBD that was significantly higher compared topre-COVID-19 historic controls (FIG. 5). Anti-S and -RBD IgG levels werehigher than IgM or IgA. Moreover, there was greater variability in theanti-RBD than the anti-S response, but the two were positivelycorrelated (FIG. 5).

Plasma neutralizing activity was determined using HIV-1 pseudotyped withSARS-CoV-2 S protein. There was a broad range of plasma neutralizingactivity 3-14 weeks after the second vaccine dose that was similar tothat elicited by natural infection in a mild disease cohort after 1.3months and greater than the activity at 6.2 months after infection (FIG.1a ; Table 1). There was no significant difference in neutralizingactivity between Moderna and Pfizer vaccines (FIG. 1b ). Plasmaneutralizing activity was directly correlated to anti-S and -RBD bindingtiters in ELISAs (FIGS. 1c-d and FIGS. 6a-d ). Finally, RBD and Sbinding and neutralizing activities were directly correlated to the timebetween receiving the first dose and blood donation with significantlylower levels in all three measurements with time (FIGS. 1e-g , and FIGS.6e-h ).

To determine whether plasma from vaccinated individuals can neutralizecirculating SARS-CoV-2 variants of concern and mutants that arise invitro under antibody pressure, w vaccinee plasma was tested against apanel of 10 mutant pseudotype viruses, including recently-reported N501Y(B1.1.7 variant), K417N, E484K and the combination of these 3 RBDmutations (501Y.V2 variant). Vaccinee plasma was significantly lesseffective in neutralizing the HIV-1 virus pseudotyped with certainSARS-CoV-2 mutant S proteins (FIGS. 1e and f and Extended Data FIG. 2).Among the volunteer plasmas tested, there was a 1 to 3, 1.3 to 2.5, and1.1 to 3 fold decrease in neutralizing activity against E484K, N501Y,and the K417N:E484K:N501Y combination, respectively (p=0.008, p=0.003,and p=0.002 respectively, FIGS. 1h and i ). It was concluded that theplasma neutralizing activity elicited by mRNA vaccination is variablybut significantly less effective against particular RBD mutants in thetested panel.

Vaccine-Elicited SARS-CoV-2 RBD-Specific Monoclonal Antibodies

Although circulating antibodies derived from plasma cells wane overtime, long-lived immune memory can persist in expanded clones of memoryB cells. Flow cytometry was used to enumerate the circulating SARS-CoV-2RBD-specific memory B cells elicited by mRNA immunization (FIG. 2a andFIGS. 7a-b ). Notably, the percentage of RBD-binding memory B cells invaccinees was significantly greater than in naturally infectedindividuals assayed after 1.3 months, but similar to the sameindividuals assayed after 6.2 months (FIG. 2b ). The percentage ofRBD-binding memory B cells in vaccinees was not correlated to the timeafter vaccination FIG. 7c ). Thus, mRNA vaccination elicits a robustSARS-CoV-2 RBD-specific B cell memory response that resembles naturalinfection.

To examine the nature of the antibodies produced by memory B cells inresponse to vaccination, 1,409 paired antibody heavy and light chainswere obtained from RBD binding single B cells from 14 individuals (n=10Moderna and n=4 Pfizer vaccinees) (Table 2). Expanded clones of cellscomprised 4-50% of the overall RBD binding memory B cell compartment(FIGS. 2-d and FIG. 7d ). Similar to natural infection, IGVH 3-53, and3-30 and some IGVL genes were significantly over-represented in theRBD-binding memory B cell compartment of vaccinated individuals (FIG. 2eand FIG. 8a ). In addition, antibodies that share the same combinationof IGHV and IGLV genes in vaccinees comprised 39% of all the clonalsequences (FIG. 8b ) and 59% when combined with naturally infectedindividuals (FIG. 2f ), and some of these antibodies were nearlyidentical (Tables 2 and 3). The number of V gene nucleotide mutations invaccinees is greater than in naturally infected individuals assayedafter 1.3 months, but lower than that in the same individuals assayedafter 6.2 months (FIG. 2g and FIG. 9a ). The length of the IgH CDR3 wassimilar in both naturally infected individuals and vaccinees, andhydrophobicity was below average (FIG. 2h and FIGS. 9a-b ). Thus, theIgG memory response is similar in individuals receiving the Pfizer andModerna vaccines, and both are rich in recurrent and clonally expandedantibody sequences.

Eighty-four representative antibodies from 4 individuals were expressedand tested for reactivity to the RBD (Table 4). The antibodies included:(1) 58 that were randomly selected from those that appeared only once,and (2) 26 representatives of expanded clones. Of the antibodies tested99% (83 out of the 84) bound to RBD, indicating that single-cell sortingby flow cytometry efficiently identified B cells producing anti-RBDantibodies (FIGS. 10a-b ; Table 4). In anti-RBD ELISAs, the meanhalf-maximal effective concentration (EC₅₀) was higher than thatobserved in infected individuals after 6 months but not significantlydifferent from antibodies obtained 1.3 months after infection (FIG. 6a ;Table 4). To examine memory B cell antibodies for binding to circulatingSARS-CoV-2 variants and antibody resistant mutants, ELISA assays wereperformed using mutant RBDs. Twenty-two (26%) of the antibodies showedat least 5-fold decreased binding to at least one of the mutant RBDs(FIGS. 10c-l ; Table 4).

SARS-CoV-2 S pseudotyped viruses were used to measure the neutralizingactivity of all 84 antibodies (FIG. 3a , Table 4). Consistent with theplasma neutralization results, the geometric mean neutralizationhalf-maximal inhibitory concentration of the vaccinee antibodies(IC₅₀=151 ng/ml) was not significantly different from antibodycollections obtained from naturally infected individuals 1.3 or 6.2months after infection (FIG. 3a ).

To examine the neutralizing breadth of the monoclonal antibodies andbegin to map their target epitopes, the 17 most potent antibodies weretested, 8 of which carried IgHV3-53, against a panel of 12 SARS-CoV-2variants: A475V is resistant to class 1 antibodies (structurally definedas described (Barnes, C. O. et al. Nature 588, 682-687 (2020)); E484Kand Q493R are resistant to class 2 antibodies; while R346S, N439K, andN440K are resistant to class 3 antibodies. Additionally, K417N, Y453F,S477R, N501Y, D614G, and R683G represent circulating variants some ofwhich have been associated with rapidly increasing case numbers. Basedon their neutralizing activity against the variants, all but 3 of theantibodies were provisionally assigned to a defined antibody class or acombination (FIG. 3b ). As seen in natural infection, a majority of theantibodies tested (9/17) were at least ten-fold less effective againstpseudotyped viruses carrying the E484K mutation. In addition, 5 of theantibodies were less potent against K417N and 4 against N501Y byten-fold or more (FIG. 3b ).

To determine whether antibody-imposed selection pressure could alsodrive the emergence of resistance mutations in vitro, an rVSV/SARS-CoV-2recombinant virus was cultured in the presence of each of 18neutralizing monoclonal antibodies. All of the tested antibodies wereselected for RBD mutations. Moreover, in all cases, the selectedmutations corresponded to residues in the binding sites of theirpresumptive antibody class (FIG. 3c ). For example, antibody 627, whichwas assigned to class 2 based on sensitivity to E484K mutation, wasselected for the emergence of the E484K mutation in vitro (FIGS. 3b andc ). Notably, 6 of the antibodies selected for K417N E or T, 5 selectedfor E484K and three selected for N501Y, T, or H, which coincide withmutations present in the circulating B1.1.17, 501Y.V2 and B1.1.28/501.V3variants that have been associated with rapidly increasing case numbersin particular locales.

Antibody Epitopes Cryo-EM Mapping of Antibody Epitopes

To further characterize antibody epitopes and mechanisms ofneutralization, seven complexes between mAb Fab fragments and theprefusion, stabilized ectodomain trimer of SARS-CoV-2 S glycoproteinwere characterized (Hsieh et al. Science: Vol. 369, Issue 6510, pp.1501-1505 (2020)) using single-particle cryo-EM (FIG. 4). Fab-Scomplexes exhibited multiple RBD-binding orientations recognizing either‘up’/‘down’ (FIGS. 4a-j ) or solely ‘up’ (FIGS. 4k-n ) RBDconformations, consistent with structurally defined antibody classesfrom natural infection. The majority of mAbs characterized (6/7)recognized epitopes that included RBD residues involved in ACE2recognition, suggesting a neutralization mechanism that directly blocksACE2-RBD interactions. Additionally, structurally defined antibodyepitopes were consistent with RBD positions that were selected inrVSV/SARS-CoV-2 recombinant virus outgrowth experiments, including K417,N439/N440, E484, and N501 (FIGS. 3c and FIGS. 4f-j, 4m-n ). Takentogether, these data indicate that functionally similar antibodies areraised during vaccination and natural infection, and that the RBDs ofspike trimers translated from the mRNA delivered by vaccination adopt asimilar distribution of ‘up’/‘down’ conformations as observed onSARS-CoV-2 virions.

Discussion

The mRNA-based SARS-CoV-2 vaccines are safe and effective and are beingdeployed globally to prevent infection and disease. The vaccine elicitsantibody responses against the RBD, the major target of neutralizingantibodies in a manner that resembles natural infection. Notably, theneutralizing antibodies produced by mRNA vaccination target the sameepitopes as natural infection. The data is consistent with SARS-CoV-2spike trimers translated from the injected RNA adopting the range ofconformations available to spikes on the surfaces of virions. Moreover,different individuals immunized with either the Moderna (mRNA-1273) orPfizer-BioNTech (BNT162b2) vaccines produce closely related andnearly-identical antibodies.

Human neutralizing monoclonal antibodies to the SARS-CoV-2 RBD can becategorized as belonging to 4 different classes based on their targetregions on the RBD. Class 1 and 2 antibodies are among the most potentand also the most abundant antibodies. These antibodies target epitopesthat overlap or are closely associated with RBD residues K417, E484, andN501. They are frequently sensitive to mutations in these residues andselect for K417N, E484K, and N501Y mutations in both yeast and VSVexpressing SARS-CoV-2 S proteins. To avert selection and escape,antibody therapies should be composed of combinations of antibodies thattarget non-overlapping epitopes.

A number of circulating SARS-CoV-2 variants have been associated withrapidly increasing case numbers and have particular prevalence in the UK(B1.1.7/501Y.V1) and South Africa (501Y. V2), and Brazil(B1.1.28/501.V3). The experiments indicate that these variants, andpotentially others that carry K417N/T, E484K, and N501Y mutations, canreduce the neutralization potency of vaccinee plasma. The comparativelymodest effects of the mutations on viral sensitivity to plasma reflectthe polyclonal nature of the neutralizing antibodies in vaccinee plasma.Nevertheless, emergence of these particular variants is consistent withthe dominance of class 1 and 2 antibody response in infected orvaccinated individuals and raises the possibility that they emerged inresponse to immune selection in individuals with suboptimal immunity.The data indicate that SARS-CoV-2 vaccines may need to be updated andimmunity monitored in order to compensate for viral evolution.

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The foregoing examples and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thescope of the invention, and all such variations are intended to beincluded within the scope of the following claims. All references citedherein are incorporated by reference in their entireties.

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220227844A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

What is claimed is:
 1. An isolated anti-SARS-CoV-2 antibody orantigen-binding fragment thereof that binds specifically to a SARS-CoV-2antigen.
 2. The antibody or antigen-binding fragment thereof of claim 1,wherein the SARS-CoV-2 antigen comprises a Spike (S) polypeptide,preferably wherein the S polypeptide is an S polypeptide of a human oran animal SARS-CoV-2, preferably wherein the SARS-CoV-2 antigencomprises the receptor-binding domain (RBD) of the S polypeptide, andfurther preferably wherein the RBD comprises amino acids 319-541 of theS-polypeptide.
 3. The antibody or antigen-binding fragment thereof ofclaim 1, wherein the antibody or antigen-binding fragment thereof iscapable of neutralizing a plurality of SARS-CoV-2 strains.
 4. Theantibody or antigen-binding fragment thereof of claim 1, comprising:three heavy chain complementarity determining regions (HCDRs) (HCDR1,HCDR2, and HCDR3) of a heavy chain variable region having the amino acidsequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159, 161, 163, 165, or 167; and three light chain CDRs (LCDR1,LCDR2, and LCDR3) of a light chain variable region having the amino acidsequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, or
 168. 5. The antibody or antigen-bindingfragment thereof of claim 1, comprising: a heavy chain variable regionhaving an amino acid sequence with at least 75% identity to the aminoacid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95,97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159, 161, 163, 165, or 167; or having the amino acid sequenceof SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, or 167; and a light chain variable region having anamino acid sequence with at least 75% identity to the amino acidsequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, or 168; or having the amino acid sequenceof SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166, or
 168. 6. The antibody or antigen-bindingfragment thereof of claim 1, comprising a heavy chain variable regionand a light chain variable region comprise the respective amino acidsequences of SEQ ID NOs: 1-2, 3-4, 5-6, 7-8, 9-10, 11-12, 13-14, 15-16,17-18, 19-20, 21-22, 23-24, 25-26, 27-28, 29-30, 31-32, 33-34, 35-36,37-38, 39-40, 41-42, 43-44, 45-46, 47-48, 49-50, 51-52, 53-54, 55-56,57-58, 59-60, 61-62, 63-64, 65-66, 67-68, 69-70, 71-72, 73-74, 75-76,77-78, 79-80, 81-82, 83-84, 85-86, 87-88, 89-90, 91-92, 93-94, 95-96,97-98, 99-100, 101-102, 103-104, 105-106, 107-108, 109-110, 111-112,113-114, 115-116, 117-118, 119-120, 121-122, 123-124, 125-126, 127-128,129-130, 131-132, 133-134, 135-136, 137-138, 139-140, 141-142, 143-144,145-146, 147-148, 149-150, 151-152, 153-154, 155-156, 157-158, 159-160,161-162, 163-164, 165-166, or 167-168.
 7. The antibody orantigen-binding fragment thereof of claim 1, wherein the antibody orantigen-binding fragment thereof is a multivalent antibody comprising(a) a first target binding site that specifically binds to an epitopewithin the S polypeptide, and (b) a second target binding site thatbinds to an epitope on a different epitope on the S polypeptide or adifferent molecule.
 8. The antibody or antigen-binding fragment thereofof claim 7, wherein the multivalent antibody is a bivalent or bispecificantibody.
 9. The antibody or the antigen-binding fragment thereof ofclaim 1, further comprising an Fc region or a variant Fc region.
 10. Theantibody or antigen-binding fragment thereof of claim 1, wherein theantibody is a monoclonal antibody.
 11. The antibody or antigen-bindingfragment thereof of claim 1, wherein the antibody is a chimericantibody, a humanized antibody, or humanized monoclonal antibody. 12.The antibody or antigen-binding fragment thereof of claim 1, wherein theantibody is a single-chain antibody, a Fab fragment, or a Fab2 fragment.13. A pharmaceutical composition comprising the antibody orantigen-binding fragment thereof of claim 1 and optionally apharmaceutically acceptable carrier or excipient.
 14. The pharmaceuticalcomposition of claim 13, wherein the pharmaceutical comprises two ormore of the antibody or antigen-binding fragment.
 15. The pharmaceuticalcomposition of claim 13, further comprising a second therapeutic agent.16. The pharmaceutical composition of claim 15, wherein the secondtherapeutic agent comprises an anti-inflammatory drug or an antiviralcompound.
 17. Use of the pharmaceutical composition of claim 13 in thepreparation of a medicament for the diagnosis, prophylaxis, treatment,or combination thereof of a condition resulting from a SARS-CoV-2infection.
 18. A nucleic acid molecule encoding a polypeptide chain ofthe antibody or antigen-binding fragment thereof of claim
 1. 19. Avector comprising the nucleic acid molecule of claim
 18. 20. A culturedhost cell comprising the nucleic acid of claim
 18. 21. A method ofpreparing an antibody, or antigen-binding portion thereof, comprising:obtaining the cultured host cell of claim 20; culturing the culturedhost cell in a medium under conditions permitting expression of apolypeptide encoded by the vector and assembling of an antibody orfragment thereof; and purifying the antibody or fragment from thecultured cell or the medium of the cell.
 22. A kit comprising apharmaceutically acceptable dose unit of the antibody or antigen-bindingfragment thereof of claim
 1. 23. A kit for the diagnosis, prognosis ormonitoring the treatment of SARS-CoV-2 infection in a subject,comprising: the antibody or antigen-binding fragment thereof of claim 1;and a least one detection reagent that binds specifically to theantibody or antigen-binding fragment thereof.
 24. A method ofneutralizing SARS-CoV-2 in a subject, comprising administering to asubject in need thereof a therapeutically effective amount of theantibody or antigen-binding fragment thereof of claim
 1. 25. A method ofpreventing or treating a SARS-CoV-2 infection, comprising administeringto a subject in need thereof a therapeutically effective amount of theantibody or antigen-binding fragment thereof of claim
 1. 26. A method ofneutralizing SARS-CoV-2 in a subject, comprising administering to asubject in need thereof a therapeutically effective amount of a firstantibody or antigen-binding fragment thereof and a second antibody orantigen-binding fragment thereof of claim 1, wherein the first antibodyor antigen-binding fragment thereof and the second antibody or antigenbinding fragment thereof exhibit synergistic activity.
 27. A method ofpreventing or treating a SARS-CoV-2 infection, comprising administeringto a subject in need thereof a therapeutically effective amount of afirst antibody or antigen-binding fragment thereof and a second antibodyor antigen-binding fragment thereof of claim 1, wherein the firstantibody or antigen-binding fragment thereof and the second antibody orantigen binding fragment thereof exhibit synergistic activity.
 28. Themethod of claim 25, further comprising administering to the subject atherapeutically effective amount of a second therapeutic agent ortherapy.
 29. The method of claim 26, wherein the first antibody orantigen-binding fragment thereof is administered before, after, orconcurrently with the second antibody or antigen-binding fragmentthereof.
 30. The method of claim 28, wherein the second therapeuticagent comprises an anti-inflammatory drug or an antiviral compound. 31.The method of claim 24, wherein the antibody or antigen-binding fragmentthereof is administered to the subject intravenously, subcutaneously, orintraperitoneally.
 32. The method of claim 24, wherein the antibody orantigen-binding fragment thereof is administered prophylactically ortherapeutically.
 33. A method for detecting the presence of SARS CoV-2in a sample comprising: contacting a sample with the antibody orantigen-binding fragment thereof of claim 1; and determining binding ofthe antibody or antigen-binding fragment to one or more SARS CoV-2antigens, wherein binding of the antibody to the one or more SARS CoV-2antigens is indicative of the presence of SARS CoV-2 in the sample.